42. Cross, DJ; Huber, BR; Silverman, MA; Cline, MM; Gill, TB; Cross, CG; Cook, DG; Minoshima, S. (2021) Intranasal Paclitaxel Alters Alzheimer's Disease Phenotypic Features in 3xTg-AD Mice.J. Alzheimers Dis. 83: 379-394 Intranasal Paclitaxel Alters Alzheimer's Disease Phenotypic Features in 3xTg-AD Mice
Alzheimer's disease; axonal transport; cognitive impairment; intranasal drug administration; microtubule stabilization
Background: Microtubule stabilizing drugs, commonly used as anti-cancer therapeutics, have been proposed for treatment of Alzheimer's disease (AD); however, many do not cross the blood-brain barrier. Objective: This research investigated if paclitaxel (PTX) delivered via the intranasal (IN) route could alter the phenotypic progression of AD in 3xTg-AD mice. Methods: We administered intranasal PTX in 3XTg-AD mice (3xTg-AD n = 15, 10 weeks and n = 10, 44 weeks, PTX: 0.6 mg/kg or 0.9%saline (SAL)) at 2-week intervals. After treatment, 3XTg-AD mice underwent manganese-enhanced magnetic resonance imaging to measure in vivo axonal transport. In a separate 3XTg-AD cohort, PTX-treated mice were tested in a radial water tread maze at 52 weeks of age after four treatments, and at 72 weeks of age, anxiety was assessed by an elevated-plus maze after 14 total treatments. Results: PTX increased axonal transport rates in treated 3XTg-AD compared to controls (p <= 0.003). Further investigation using an in vitro neuron model of A beta-induced axonal transport disruption confirmed PTX prevented axonal transport deficits. Confocal microscopy after treatment found fewer phospho-tau containing neurons (5.25 +/- 3.8 versus 8.33 +/- 2.5, p < 0.04) in the CA1, altered microglia, and reduced reactive astrocytes. PTX improved performance of 3xTg-AD on the water tread maze compared to controls and not significantly different from WT (Day 5, 143.8 +/- 43 versus 91.5 +/- 77s and Day 12, 138.3 +/- 52 versus 107.7 +/- 75s for SAL versus PTX). Elevated plus maze revealed that PTX-treated 3xTg-AD mice spent more time exploring open arms (Open arm 129.1 +/- 80 versus 20.9 +/- 31s for PTX versus SAL, p <= 0.05). Conclusion: Taken collectively, these findings indicate that intranasal-administered microtubule-stabilizing drugs may offer a potential therapeutic option for treating AD. DOI PubMed
41. Gan, KJ; Akram, A; Blasius, TL; Ramser, EM; Budaitis, BG; Gabrych, DR; Verhey, KJ; Silverman, MA. (2020) GSK3 beta Impairs KIF1A Transport in a Cellular Model of Alzheimer's Disease but Does Not Regulate Motor Motility at S402.eNeuro 7 GSK3 beta Impairs KIF1A Transport in a Cellular Model of Alzheimer's Disease but Does Not Regulate Motor Motility at S402
Alzheimer's disease; amyloid beta oligomers; axonal transport; glycogen synthase kinase beta; kinesin-3 (KIF1A); motor protein phosphorylation
Impairment of axonal transport is an early pathologic event that precedes neurotoxicity in Alzheimer's disease (AD). Soluble amyloid-beta oligomers (A beta Os), a causative agent of AD, activate intracellular signaling cascades that trigger phosphorylation of many target proteins, including tau, resulting in microtubule destabilization and transport impairment. Here, we investigated how KIF1A, a kinesin-3 family motor protein required for the transport of neurotrophic factors, is impaired in mouse hippocampal neurons treated with A beta Os. By live cell imaging, we observed that A beta Os inhibit transport of KIF1A-GFP similarly in wild-type and tau knock-out neurons, indicating that tau is not required for this effect. Pharmacological inhibition of glycogen synthase kinase 3 beta (GSK3 beta), a kinase overactivated in AD, prevented the transport defects. By mass spectrometry on KIF1A immunoprecipitated from transgenic AD mouse brain, we detected phosphorylation at S402, which conforms to a highly conserved GSK3 beta consensus site. We confirmed that this site is phosphorylated by GSK3 beta in vitro. Finally, we tested whether a phosphomimic of S402 could modulate KIF1A motility in control and A beta O-treated mouse neurons and in a Golgi dispersion assay devoid of endogenous KIF1A. In both systems, transport driven by mutant motors was similar to that of WT motors. In conclusion, GSK3 beta impairs KIF1A transport but does not regulate motor motility at S402. Further studies are required to determine the specific phosphorylation sites on KIF1A that regulate its cargo binding and/or motility in physiological and disease states. DOI PubMed
40. Gabrych, DR; Lau, VZ; Niwa, S; Silverman, MA. (2019) Going Too Far Is the Same as Falling Short(dagger): Kinesin-3 Family Members in Hereditary Spastic Paraplegia.Front. Cell. Neurosci. 13 Going Too Far Is the Same as Falling Short(dagger): Kinesin-3 Family Members in Hereditary Spastic Paraplegia
KIF1; axonal transport; hereditary spastic paraplegia (HSP); neurodegenarative disease; vesicle trafficking; kinesin
Proper intracellular trafficking is essential for neuronal development and function, and when any aspect of this process is dysregulated, the resulting "transportopathy" causes neurological disorders. Hereditary spastic paraplegias (HSPs) are a family of such diseases attributed to over 80 spastic gait genes (SPG), specifically characterized by lower extremity spasticity and weakness. Multiple genes in the trafficking pathway such as those relating to microtubule structure and function and organelle biogenesis are representative disease loci. Microtubule motor proteins, or kinesins, are also causal in HSP, specifically mutations in Kinesin-I/KIF5A (SPG10) and two kinesin-3 family members; KIF1A (SPG30) and KIF1C (SPG58). KIF1A is a motor enriched in neurons, and involved in the anterograde transport of a variety of vesicles that contribute to pre- and post-synaptic assembly, autophagic processes, and neuron survival. KIF1C is ubiquitously expressed and, in addition to anterograde cargo transport, also functions in retrograde transport between the Golgi and the endoplasmic reticulum. Only a handful of KIF1C cargos have been identified; however, many have crucial roles such as neuronal differentiation, outgrowth, plasticity and survival. HSP-related kinesin-3 mutants are characterized mainly as loss-of-function resulting in deficits in motility, regulation, and cargo binding. Gain-of-function mutants are also seen, and are characterized by increased microtubule-on rates and hypermotility. Both sets of mutations ultimately result in misdelivery of critical cargos within the neuron. This likely leads to deleterious cell biological cascades that likely underlie or contribute to HSP clinical pathology and ultimately, symptomology. Due to the paucity of histopathological or cell biological data assessing perturbations in cargo localization, it has been difficult to positively link these mutations to the outcomes seen in HSPs. Ultimately, the goal of this review is to encourage future academic and clinical efforts to focus on "transportopathies" through a cargo-centric lens. DOI PubMed
39. Gomes, LMF; Mahammed, A; Prosser, KE; Smith, JR; Silverman, MA; Walsby, CJ; Gross, Z; Storr, T. (2019) A catalytic antioxidant for limiting amyloid-beta peptide aggregation and reactive oxygen species generation.Chem. Sci. 10 A catalytic antioxidant for limiting amyloid-beta peptide aggregation and reactive oxygen species generation
Alzheimer's disease (AD) is a multifaceted disease that is characterized by increased oxidative stress, metal-ion dysregulation, and the formation of intracellular neurofibrillary tangles and extracellular amyloid-beta (A beta) aggregates. In this work we report the large affinity binding of the iron(III) 2,17-bis-sulfonato-5,10,15-tris(pentafluorophenyl)corrole complex FeL1 to the A beta peptide (K-d similar to 10(-7)) and the ability of the bound FeL1 to act as a catalytic antioxidant in both the presence and absence of Cu(II) ions. Specific findings are that: (a) an A beta histidine residue binds axially to FeL1; (b) that the resulting adduct is an efficient catalase; (c) this interaction restricts the formation of high molecular weight peptide aggregates. UV-Vis and electron paramagnetic resonance (EPR) studies show that although the binding of FeL1 does not influence the A beta-Cu(II) interaction (K-d similar to 10(-10)), bound FeL1 still acts as an antioxidant thereby significantly limiting reactive oxygen species (ROS) generation from A beta-Cu. Overall, FeL1 is shown to bind to the A beta peptide, and modulate peptide aggregation. In addition, FeL1 forms a ternary species with A beta-Cu(II) and impedes ROS generation, thus showing the promise of discrete metal complexes to limit the toxicity pathways of the A beta peptide. DOI PubMed
38. Julien, C; Tomberlin, C; Roberts, CM; Akram, A; Stein, GH; Silverman, MA; Link, CD. (2018) In vivo induction of membrane damage by beta-amyloid peptide oligomers.Acta Neuropathol. Commun. 6 In vivo induction of membrane damage by beta-amyloid peptide oligomers
Alzheimer's disease; beta-amyloid; Tau; Caenorhabditis elegans; Pore-forming toxin
Exposure to the -amyloid peptide (A) is toxic to neurons and other cell types, but the mechanism(s) involved are still unresolved. Synthetic A oligomers can induce ion-permeable pores in synthetic membranes, but whether this ability to damage membranes plays a role in the ability of A oligomers to induce tau hyperphosphorylation, or other disease-relevant pathological changes, is unclear. To examine the cellular responses to A exposure independent of possible receptor interactions, we have developed an in vivo C. elegans model that allows us to visualize these cellular responses in living animals. We find that feeding C. elegans E. coli expressing human A induces a membrane repair response similar to that induced by exposure to the CRY5B, a known pore-forming toxin produced by B. thuringensis. This repair response does not occur when C. elegans is exposed to an A Gly37Leu variant, which we have previously shown to be incapable of inducing tau phosphorylation in hippocampal neurons. The repair response is also blocked by loss of calpain function, and is altered by loss-of-function mutations in the C. elegans orthologs of BIN1 and PICALM, well-established risk genes for late onset Alzheimer's disease. To investigate the role of membrane repair on tau phosphorylation directly, we exposed hippocampal neurons to streptolysin O (SLO), a pore-forming toxin that induces a well-characterized membrane repair response. We find that SLO induces tau hyperphosphorylation, which is blocked by calpain inhibition. Finally, we use a novel biarsenical dye-tagging approach to show that the Gly37Leu substitution interferes with A multimerization and thus the formation of potentially pore-forming oligomers. We propose that A-induced tau hyperphosphorylation may be a downstream consequence of induction of a membrane repair process. DOI PubMed
37. Zhu, YP; Shan, XY; Safarpour, F; Go, NE; Li, N; Shan, A; Huang, MNC; Deen, M; Holicek, V; Ashmus, R; Madden, Z; Gorski, S; Silverman, MA; Vocadlo, DJ. (2018) Pharmacological Inhibition of O-GIcNAcase Enhances Autophagy in Brain through an mTOR-Independent Pathway.ACS Chem. Neurosci. 9 Pharmacological Inhibition of O-GIcNAcase Enhances Autophagy in Brain through an mTOR-Independent Pathway
Alzheimer's disease; glycosylation; autophagy; neurodegeneration; O-GlcNAc; Thiamet-G
The glycosylation of nucleocytoplasmic proteins with O-linked N-acetylglucosamine residues (O-GlcNAc) is conserved among metazoans and is particularly abundant within brain. O-GlcNAc is involved in diverse cellular processes ranging from the regulation of gene expression to stress response. Moreover, O-GlcNAc is implicated in various diseases including cancers, diabetes, cardiac dysfunction, and neurodegenerative diseases. Pharmacological inhibition of O-GlcNAcase (OGA), the sole enzyme that removes O-GlcNAc, reproducibly slows neurodegeneration in various Alzheimer's disease (AD) mouse models manifesting either tau or amyloid pathology. These data have stimulated interest in the possibility of using OGA-selective inhibitors as pharmaceuticals progression of AD. The mechanisms mediating the neuroprotective effects of OGA inhibitors, however, remain poorly understood. Here we show, using a range of methods in neuroblastoma N2a cells, in primary rat neurons, and in mouse brain, that selective OGA inhibitors stimulate autophagy through an mTOR-independent pathway without obvious toxicity. Additionally, OGA inhibition significantly decreased the levels of toxic protein species associated with AD pathogenesis in the JNPL3 tauopathy mouse model as well as the 3XTg-AD mouse model. These results strongly suggest that OGA inhibitors act within brain through a mechanism involving enhancement of autophagy, which aids the brain in combatting the accumulation of toxic protein species. Our study supports OGA inhibition being a feasible. therapeutic strategy for hindering the progression of AD and other neurodegenerative diseases. Moreover, these data suggest more targeted strategies to stimulate autophagy in an mTOR-independent manner may be found within the O-GIcNAc pathway. These findings should aid the advancement of OGA inhibitors within the clinic. DOI PubMed
36. Gan, KJ; Silverman, MA. (2016) Imaging organelle transport in primary hippocampal neurons treated with amyloid-beta oligomers.Methods Cell Biol. 131: 425-451 Imaging organelle transport in primary hippocampal neurons treated with amyloid-beta oligomers
We describe a strategy for fluorescent imaging of organelle transport in primary hippocampal neurons treated with amyloid-beta (A beta) peptides that cause Alzheimer's disease (AD). This method enables careful, rigorous analyses of axonal transport defects, which are implicated in AD and other neurodegenerative diseases. Moreover, we present and emphasize guidelines for investigating A beta-induced mechanisms of axonal transport disruption in the absence of nonspecific, irreversible cellular toxicity. This approach should be accessible to most laboratories equipped with cell culture facilities and a standard fluorescent microscope and may be adapted to other cell types. DOI PubMed
35. Robinson, BJ; Stanisavljevic, B; Silverman, MA; Scalettar, BA. (2016) Stochastic Subcellular Organization of Dense-Core Vesicles Revealed by Point Pattern Analysis.Biophysical Journal 111: 852-863 Stochastic Subcellular Organization of Dense-Core Vesicles Revealed by Point Pattern Analysis
Dense-core vesicles (DCVs) are regulated secretory organelles found in many types of neurons. In neurons of the hippocampus, their cargo includes proteins that mediate several pivotal processes, including differentiation and synaptic plasticity. Motivated by interest in DCV distribution and its impact on cargo action, we have used fluorescence microscopy and statistical analysis to develop a quantitative model of the subcellular organization of DCVs in hippocampal neurons that are spontaneously active (their most prevalent state). We also have tested the functionally motivated hypothesis that these organelles are synaptically enriched. Variance-to-mean ratio, frequency distribution, and Moran's autocorrelation analyses reveal that DCV distribution along shafts, and within synapses, follows Poisson statistics, establishing that stochastically dictated organization sustains cargo function. Occupancy in boutons exceeds that at nearby extrasynaptic axonal sites by approximately threefold, revealing significant local presynaptic enrichment. Widespread stochastic organization is consistent with the emerging functional importance of synaptically and extrasynaptically localized DCVs. Presynaptic enrichment is consistent with the established importance of protecting presynaptic sites from depletion of DCV cargo. These results enhance understanding of the link between DCV organization and mechanisms of cargo action, and they reinforce the emerging theme that randomness is a prevalent aspect of synaptic organization and composition. DOI
34. Cavolo, SL; Zhou, CM; Ketcham, SA; Suzuki, MM; Ukalovic, K; Silverman, MA; Schroer, TA; Levitan, ES. (2015) Mycalolide B dissociates dynactin and abolishes retrograde axonal transport of dense-core vesicles.Molecular Biology of the Cell 26: 2664-2672 Mycalolide B dissociates dynactin and abolishes retrograde axonal transport of dense-core vesicles
Axonal transport is critical for maintaining synaptic transmission. Of interest, anterograde and retrograde axonal transport appear to be interdependent, as perturbing one directional motor often impairs movement in the opposite direction. Here live imaging of Drosophila and hippocampal neuron dense-core vesicles (DCVs) containing a neuropeptide or brain-derived neurotrophic factor shows that the F-actin depolymerizing macrolide toxin mycalolide B (MB) rapidly and selectively abolishes retrograde, but not anterograde, transport in the axon and the nerve terminal. Latrunculin A does not mimic MB, demonstrating that F-actin depolymerization is not responsible for unidirectional transport inhibition. Given that dynactin initiates retrograde transport and that amino acid sequences implicated in macrolide toxin binding are found in the dynactin component actin-related protein 1, we examined dynactin integrity. Remarkably, cell extract and purified protein experiments show that MB induces disassembly of the dynactin complex. Thus imaging selective retrograde transport inhibition led to the discovery of a small-molecule dynactin disruptor. The rapid unidirectional inhibition by MB suggests that dynactin is absolutely required for retrograde DCV transport but does not directly facilitate ongoing anterograde DCV transport in the axon or nerve terminal. More generally, MB's effects bolster the conclusion that anterograde and retrograde axonal transport are not necessarily interdependent. DOI
33. Gan, KJ; Morihara, T; Silverman, MA. (2015) Atlas stumbled: Kinesin light chain-1 variant E triggers a vicious cycle of axonal transport disruption and amyloid-beta generation in Alzheimer's disease.Bioessays 37: 131-141 Atlas stumbled: Kinesin light chain-1 variant E triggers a vicious cycle of axonal transport disruption and amyloid-beta generation in Alzheimer's disease
alternative splicing; Alzheimer's disease; amyloid-beta; amyloid precursor protein; axonal transport; kinesin; kinesin light chain
Substantial evidence implicates fast axonal transport (FAT) defects in neurodegeneration. In Alzheimer's disease (AD), it is controversial whether transport defects cause or arise from amyloid-beta (A beta)-induced toxicity. Using a novel, unbiased genetic screen, Morihara et al. identified kinesin light chain-1 splice variant E (KLC1vE) as a modifier of A beta accumulation. Here, we propose three mechanisms to explain this causal role. First, KLC1vE reduces APP transport, leading to A beta accumulation. Second, reduced transport of APP by KLC1vE triggers an ER stress response that activates the amyloidogenic pathway. Third, KLC1vE impairs transport of other KLC1 cargos that regulate amyloidogenesis, promoting Ab retention within the secretory pathway. Collectively, KLC1vE perpetuates a vicious cycle of A beta generation, kinase dysregulation, and global FAT impairment that inevitably leads to cellular toxicity. These concepts implicate alternative splicing of KLC1 in AD and suggest that the reciprocal influence of transport mechanisms on disease states contributes to neurodegeneration. DOI PubMed
32. Gan, KJ; Silverman, MA. (2015) Dendritic and axonal mechanisms of Ca2+ elevation impair BDNF transport in A beta oligomer-treated hippocampal neurons.Molecular Biology of the Cell 26: 1058-1071 Dendritic and axonal mechanisms of Ca2+ elevation impair BDNF transport in A beta oligomer-treated hippocampal neurons
Disruption of fast axonal transport (FAT) and intracellular Ca2+ dysregulation are early pathological events in Alzheimer's disease (AD). Amyloid-beta oligomers (A beta Os), a causative agent of AD, impair transport of BDNF independent of tau by nonexcitotoxic activation of calcineurin (CaN). Ca2+-dependent mechanisms that regulate the onset, severity, and spatiotemporal progression of BDNF transport defects from dendritic and axonal A beta O binding sites are unknown. Here we show that BDNF transport defects in dendrites and axons are induced simultaneously but exhibit different rates of decline. The spatiotemporal progression of FAT impairment correlates with Ca2+ elevation and CaN activation first in dendrites and subsequently in axons. Although many axonal pathologies have been described in AD, studies have primarily focused only on the dendritic effects of A beta Os despite compelling reports of presynaptic A beta Os in AD models and patients. Indeed, we observe that dendritic CaN activation converges on Ca2+ influx through axonal voltage-gated Ca2+ channels to impair FAT. Finally, FAT defects are prevented by dantrolene, a clinical compound that reduces Ca2+ release from the ER. This work establishes a novel role for Ca2+ dysregulation in BDNF transport disruption and tau-independent A beta toxicity in early AD. DOI PubMed
31. Takach, O; Gill, TB; Silverman, MA. (2015) Modulation of insulin signaling rescues BDNF transport defects independent of tau in amyloid-beta oligomer-treated hippocampal neurons.Neurobiology of Aging 36: 1378-1382 Modulation of insulin signaling rescues BDNF transport defects independent of tau in amyloid-beta oligomer-treated hippocampal neurons
Axonal transport; Alzheimer's disease; Insulin signaling; GSK3beta; Brain-derived neurotrophic factor; Hippocampal neuron
Defective brain insulin signaling contributes to the cognitive deficits in Alzheimer's disease (AD). Amyloid-beta oligomers (A beta Os), the primary neurotoxin implicated in AD, downregulate insulin signaling by impairing protein kinase B/AKT, thereby overactivating glycogen synthase kinase-3 beta. By this mechanism, A beta Os may also impair axonal transport before tau-induced cytoskeletal collapse and cell death. Here, we demonstrate that a constitutively active form of protein kinase B/AKT prevents brain-derived neurotrophic factor (BDNF) transport defects in A beta O-treated primary neurons from wild type (tau(+/+)) and tau knockout (tau(-/-)) mice. Remarkably, inhibition of glycogen synthase kinase-3 beta rescues BDNF transport defects independent of tau. Furthermore, exendin-4, an anti-diabetes agent, restores normal BDNF axonal transport by stimulating the glucagon-like peptide-1 receptor to activate the insulin pathway. Collectively, our findings indicate that normalized insulin signaling can both prevent and reverse BDNF transport defects in A beta O-treated neurons. Ultimately, this work may reveal novel therapeutic targets that regulate BDNF trafficking, promote its secretion and uptake, and prolong neuronal survival during AD progression. (C) 2015 Elsevier Inc. All rights reserved. DOI PubMed
29. Morihara, T; Hayashi, N; Yokokoji, M; Akatsu, H; Silverman, MA; Kimura, N; Sato, M; Saito, Y; Suzuki, T; Yanagida, K; Kodama, TS; Tanaka, T; Okochi, M; Tagami, S; Kazui, H; Kudo, T; Hashimoto, R; Itoh, N; Nishitomi, K; Yamaguchi-Kabata, Y; Tsunoda, T; Takamura, H; Katayama, T; Kimura, R; Kaminoa, K; Hashizume, Y; Takeda, M. (2014) Transcriptome analysis of distinct mouse strains reveals kinesin light chain-1 splicing as an amyloid-beta accumulation modifier.Proceedings of the National Academy of Sciences of the United States of America 111: 2638-2643 Transcriptome analysis of distinct mouse strains reveals kinesin light chain-1 splicing as an amyloid-beta accumulation modifier
mouse-to-human translation; alternative splicing
Alzheimer's disease (AD) is characterized by the accumulation of amyloid-beta (A beta). The genes that govern this process, however, have remained elusive. To this end, we combined distinct mouse strains with transcriptomics to directly identify disease-relevant genes. We show that AD model mice (APP-Tg) with DBA/2 genetic backgrounds have significantly lower levels of A beta accumulation compared with SJL and C57BL/6 mice. We then applied brain transcriptomics to reveal the genes in DBA/2 that suppress A beta accumulation. To avoid detecting secondarily affected genes by A beta, we used non-Tg mice in the absence of A beta pathology and selected candidate genes differently expressed in DBA/2 mice. Additional transcriptome analysis of APP-Tg mice with mixed genetic backgrounds revealed kinesin light chain-1 (Klc1) as an A beta modifier, indicating a role for intracellular trafficking in A beta accumulation. A beta levels correlated with the expression levels of Klc1 splice variant E and the genotype of Klc1 in these APP-Tg mice. In humans, the expression levels of KLC1 variant E in brain and lymphocyte were significantly higher in AD patients compared with unaffected individuals. Finally, functional analysis using neuroblastoma cells showed that overexpression or knockdown of KLC1 variant E increases or decreases the production of A beta, respectively. The identification of KLC1 variant E suggests that the dysfunction of intracellular trafficking is a causative factor of A beta pathology. This unique combination of distinct mouse strains and model mice with transcriptomics is expected to be useful for the study of genetic mechanisms of other complex diseases. DOI
28. Yuzwa, SA; Shan, XY; Jones, BA; Zhao, G; Woodward, ML; Li, XJ; Zhu, YP; McEachern, EJ; Silverman, MA; Watson, NV; Gong, CX; Vocadlo, DJ. (2014) Pharmacological inhibition of O-GlcNAcase (OGA) prevents cognitive decline and amyloid plaque formation in bigenic tau/APP mutant mice.Molecular Neurodegeneration 9 Pharmacological inhibition of O-GlcNAcase (OGA) prevents cognitive decline and amyloid plaque formation in bigenic tau/APP mutant mice
tau; Amyloid precursor protein; O-GlcNAc; Thiamet-G
Background: Amyloid plaques and neurofibrillary tangles (NFTs) are the defining pathological hallmarks of Alzheimer's disease (AD). Increasing the quantity of the O-linked N-acetylglucosamine (O-GlcNAc) post-translational modification of nuclear and cytoplasmic proteins slows neurodegeneration and blocks the formation of NFTs in a tauopathy mouse model. It remains unknown, however, if O-GlcNAc can influence the formation of amyloid plaques in the presence of tau pathology. Results: We treated double transgenic TAPP mice, which express both mutant human tau and amyloid precursor protein (APP), with a highly selective orally bioavailable inhibitor of the enzyme responsible for removing O-GlcNAc (OGA) to increase O-GlcNAc in the brain. We find that increased O-GlcNAc levels block cognitive decline in the TAPP mice and this effect parallels decreased beta-amyloid peptide levels and decreased levels of amyloid plaques. Conclusions: This study indicates that increased O-GlcNAc can influence beta-amyloid pathology in the presence of tau pathology. The findings provide good support for OGA as a promising therapeutic target to alter disease progression in Alzheimer disease. DOI PubMed
26. Heydet, D; Chen, LX; Larter, CZ; Inglis, C; Silverman, MA; Farrell, GC; Leroux, MR. (2013) A truncating mutation of Alms1 reduces the number of hypothalamic neuronal cilia in obese mice.Developmental Neurobiology 73: 1-13 A truncating mutation of Alms1 reduces the number of hypothalamic neuronal cilia in obese mice
primary cilium; hypothalamus; obesity; Alms1 BARDET-BIEDL-SYNDROME; ALSTROM-SYNDROME PROTEIN; INTRAFLAGELLAR TRANSPORT; HIPPOCAMPAL-NEURONS; MONOGENIC DISORDERS; RAT HYPOTHALAMUS; SIGNALING CENTER; BASAL BODIES; DISEASE; SOMATOSTATIN
Primary cilia are ubiquitous cellular antennae whose dysfunction collectively causes various disorders, including vision and hearing impairment, as well as renal, skeletal, and central nervous system anomalies. One ciliopathy, Alstrom syndrome, is closely related to BardetBiedl syndrome (BBS), sharing amongst other phenotypic features morbid obesity. As the cellular and molecular links between weight regulation and cilia are poorly understood, we used the obese mouse strain foz/foz, bearing a truncating mutation in the Alstrom syndrome protein (Alms1), to help elucidate why it develops hyperphagia, leading to early onset obesity and metabolic anomalies. Our in vivo studies reveal that Alms1 localizes at the base of cilia in hypothalamic neurons, which are implicated in the control of satiety. Alms1 is lost from this location in foz/foz mice, coinciding with a strong postnatal reduction (70%) in neurons displaying cilia marked with adenylyl cyclase 3 (AC3), a signaling protein implicated in obesity. Notably, the reduction in AC3-bearing cilia parallels the decrease in cilia containing two appetite-regulating proteins, Mchr1 and Sstr3, as well as another established Arl13b ciliary marker, consistent with progressive loss of cilia during development. Together, our results suggest that Alms1 maintains the function of neuronal cilia implicated in weight regulation by influencing the maintenance and/or stability of the organelle. Given that Mchr1 and Sstr3 localization to remaining cilia is maintained in foz/foz animals but known to be lost from BBS knockout mice, our findings suggest different molecular etiologies for the satiety defects associated with the Alstrom syndrome and BBS ciliopathies. (C) 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2013 DOI
25. Petoukhov, E; Fernando, S; Mills, F; Shivji, F; Hunter, D; Krieger, C; Silverman, MA; Bamji, SX. (2013) Activity-dependent secretion of progranulin from synapses.J. Cell Sci. 126: 5412-5421 Activity-dependent secretion of progranulin from synapses
Progranulin; Synapses; Activity; Secretion; Hippocampal culture; Protein trafficking; Frontotemporal dementia
The secreted growth factor progranulin (PGRN) has been shown to be important for regulating neuronal survival and outgrowth, as well as synapse formation and function. Mutations in the PGRN gene that result in PGRN haploinsufficiency have been identified as a major cause of frontotemporal dementia (FTD). Here we demonstrate that PGRN is colocalized with dense-core vesicle markers and is cotransported with brain-derived neurotrophic factor (BDNF) within axons and dendrites of cultured hippocampal neurons in both anterograde and retrograde directions. We also show that PGRN is secreted in an activity-dependent manner from synaptic and extrasynaptic sites, and that the temporal profiles of secretion are distinct in axons and dendrites. Neuronal activity is also shown to increase the recruitment of PGRN to synapses and to enhance the density of PGRN clusters along axons. Finally, treatment of neurons with recombinant PGRN is shown to increase synapse density, while decreasing the size of the presynaptic compartment and specifically the number of synaptic vesicles per synapse. Together, this indicates that activity-dependent secretion of PGRN can regulate synapse number and structure. DOI PubMed
24. Ramser, EM; Gan, KJ; Decker, H; Fan, EY; Suzuki, MM; Ferreira, ST; Silverman, MA. (2013) Amyloid-beta oligomers induce tau-independent disruption of BDNF axonal transport via calcineurin activation in cultured hippocampal neurons.Mol. Biol. Cell 24: 2494-2505 Amyloid-beta oligomers induce tau-independent disruption of BDNF axonal transport via calcineurin activation in cultured hippocampal neurons
Disruption of fast axonal transport (FAT) is an early pathological event in Alzheimer's disease (AD). Soluble amyloid-beta oligomers (A beta Os), increasingly recognized as proximal neurotoxins in AD, impair organelle transport in cultured neurons and transgenic mouse models. A beta Os also stimulate hyperphosphorylation of the axonal microtubule-associated protein, tau. However, the role of tau in FAT disruption is controversial. Here we show that A beta Os reduce vesicular transport of brain-derived neurotrophic factor (BDNF) in hippocampal neurons from both wild-type and tau-knockout mice, indicating that tau is not required for transport disruption. FAT inhibition is not accompanied by microtubule destabilization or neuronal death. Significantly, inhibition of calcineurin (CaN), a calcium-dependent phosphatase implicated in AD pathogenesis, rescues BDNF transport. Moreover, inhibition of protein phosphatase 1 and glycogen synthase kinase 3 beta, downstream targets of CaN, prevents BDNF transport defects induced by A beta Os. We further show that A beta Os induce CaN activation through nonexcitotoxic calcium signaling. Results implicate CaN in FAT regulation and demonstrate that tau is not required for A beta O-induced BDNF transport disruption. DOI PubMed
23. Bomfim, TR; Forny-Germano, L; Sathler, LB; Brito-Moreira, J; Houzel, JC; Decker, H; Silverman, MA; Kazi, H; Melo, HM; McClean, PL; Holscher, C; Arnold, SE; Talbot, K; Klein, WL; Munoz, DP; Ferreira, ST; De Felice, FG. (2012) An anti-diabetes agent protects the mouse brain from defective insulin signaling caused by Alzheimer's disease-associated A beta oligomers.Journal of Clinical Investigation 122: 1339-1353 An anti-diabetes agent protects the mouse brain from defective insulin signaling caused by Alzheimer's disease-associated A beta oligomers
Defective brain insulin signaling has been suggested to contribute to the cognitive deficits in patients with Alzheimer's disease (AD). Although a connection between AD and diabetes has been suggested, a major unknown is the mechanism(s) by which insulin resistance in the brain arises in individuals with AD. Here, we show that serine phosphorylation of IRS-1 (IRS-1pSer) is common to both diseases. Brain tissue from humans with AD had elevated levels of IRS-1pSer and activated JNK, analogous to what occurs in peripheral tissue in patients with diabetes. We found that amyloid-beta peptide (A beta) oligomers, synaptotoxins that accumulate in the brains of AD patients, activated the JNK/TNF-alpha pathway, induced IRS-1 phosphorylation at multiple serine residues, and inhibited physiological IRS-1pTyr in mature cultured hippocarnpal neurons. Impaired IRS-1 signaling was also present in the hippocampi of Tg mice with a brain condition that models AD. Importantly, intracerebroventricular injection of A beta oligomers triggered hippocampal IRS-1pSer and JNK activation in cynomolgus monkeys. The oligomer-induced neuronal pathologies observed in vitro, including impaired axonal transport, were prevented by exposure to exendin-4 (exenatide), an anti-diabetes agent. In Tg mice, exendin-4 decreased levels of hippocampal IRS-1pSer and activated JNK and improved behavioral measures of cognition. By establishing molecular links between the dysregulated insulin signaling in AD and diabetes, our results open avenues for the investigation of new therapeutics in AD. DOI
22. Fonte, V; Dostal, V; Roberts, CM; Gonzales, P; Lacor, P; Magrane, J; Dingwell, N; Fan, EY; Silverman, MA; Stein, GH; Link, CD. (2011) A glycine zipper motif mediates the formation of toxic beta-amyloid oligomers in vitro and in vivo.Molecular Neurodegeneration 6 A glycine zipper motif mediates the formation of toxic beta-amyloid oligomers in vitro and in vivo
Alzheimer's disease; C. elegans; pore-forming toxin; glycine motif
Background: The beta-amyloid peptide (A beta) contains a Gly-XXX-Gly-XXX-Gly motif in its C-terminal region that has been proposed to form a "glycine zipper" that drives the formation of toxic A beta oligomers. We have tested this hypothesis by examining the toxicity of A beta variants containing substitutions in this motif using a neuronal cell line, primary neurons, and a transgenic C. elegans model. Results: We found that a Gly37Leu substitution dramatically reduced A beta toxicity in all models tested, as measured by cell dysfunction, cell death, synaptic alteration, or tau phosphorylation. We also demonstrated in multiple models that A beta Gly37Leu is actually anti-toxic, thereby supporting the hypothesis that interference with glycine zipper formation blocks assembly of toxic A beta oligomers. To test this model rigorously, we engineered second site substitutions in A beta predicted by the glycine zipper model to compensate for the Gly37Leu substitution and expressed these in C. elegans. We show that these second site substitutions restore in vivo A beta toxicity, further supporting the glycine zipper model. Conclusions: Our structure/function studies support the view that the glycine zipper motif present in the C-terminal portion of A beta plays an important role in the formation of toxic A beta oligomers. Compounds designed to interfere specifically with formation of the glycine zipper could have therapeutic potential. DOI
21. Lo, KY; Kuzmin, A; Unger, SM; Petersen, JD; Silverman, MA. (2011) KIF1A is the primary anterograde motor protein required for the axonal transport of dense-core vesicles in cultured hippocampal neurons.Neuroscience Letters 491 KIF1A is the primary anterograde motor protein required for the axonal transport of dense-core vesicles in cultured hippocampal neurons
Dense-core vesicle; Axonal transport; Neuropeptide; Hippocampal neuron; KIF1A; Bidirectional transport
Dense-core vesicles (DCVs) are responsible for transporting, processing, and secreting neuropeptide cargos that mediate a wide range of biological processes, including neuronal development, survival, and learning and memory. DCVs are synthesized in the cell body and are transported by kinesin motor proteins along microtubules to pre- and postsynaptic release sites. Due to the dependence on kinesin-based transport, we sought to determine if the kinesin-3 family member, KIF1A, transports DCVs in primary cultured hippocampal neurons, as has been described for invertebrate neurons. Two-color, live-cell imaging showed that the DCV markers, chromogranin A-RFP and BDNF-RFP, move together with KIF1A-GFP in both the anterograde and retrograde directions. To demonstrate a functional role for KIF1A in DCV transport, motor protein expression in neurons was reduced using RNA interference (shRNA). Fluorescently tagged DCV markers showed a significant reduction in organelle flux in cells expressing shRNA against KIF1A. The transport of cargo driven by motors other than KIF1A, including mitochondria and the transferrin receptor, was unaffected in KIF1A shRNA expressing cells. Taken together, these data support a primary role for KIF1A in the anterograde transport of DCVs in mammalian neurons, and also provide evidence that KIF1A remains associated with DCVs during retrograde DCV transport. Crown Copyright (C) 2011 Published by Elsevier Ireland Ltd. All rights reserved. DOI
19. Decker, H; Lo, KY; Unger, SM; Ferreira, ST; Silverman, MA. (2010) Amyloid-beta Peptide Oligomers Disrupt Axonal Transport through an NMDA Receptor-Dependent Mechanism That Is Mediated by Glycogen Synthase Kinase 3 beta in Primary Cultured Hippocampal Neurons.Journal of Neuroscience 30: 9166-9171 Amyloid-beta Peptide Oligomers Disrupt Axonal Transport through an NMDA Receptor-Dependent Mechanism That Is Mediated by Glycogen Synthase Kinase 3 beta in Primary Cultured Hippocampal Neurons
Disruption of axonal transport is a hallmark of several neurodegenerative diseases, including Alzheimer's disease (AD). Even though defective transport is considered an early pathologic event, the mechanisms by which neurodegenerative insults impact transport are poorly understood. We show that soluble oligomers of the amyloid-beta peptide (A beta Os), increasingly recognized as the proximal neurotoxins in AD pathology, induce disruption of organelle transport in primary hippocampal neurons in culture. Live imaging of fluorescent protein-tagged organelles revealed a marked decrease in axonal trafficking of dense-core vesicles and mitochondria in the presence of 0.5 mu M A beta Os. NMDA receptor (NMDAR) antagonists, including D-AP5, MK-801, and memantine, prevented the disruption of trafficking, thereby identifying signals for A beta O action at the cell membrane. Significantly, both pharmacological inhibition of glycogen synthase kinase-3 beta (GSK-3 beta) and transfection of neurons with a kinase-dead form of GSK-3 beta prevented the transport defect. Finally, we demonstrate by biochemical and immunocytochemical means that A beta Os do not affect microtubule stability, indicating that disruption of transport involves a more subtle mechanism than microtubule destabilization, likely the dysregulation of intracellular signaling cascades. Results demonstrate that A beta Os negatively impact axonal transport by a mechanism that is initiated by NMDARs and mediated by GSK-3 beta and establish a new connection between toxic A beta oligomers and AD pathology.Website
18.Silverman, MA; Kaech, S; Ramser, EM; Lu, X; Lasarev, MR; Nagalla, S; Banker, G. (2010) Expression of Kinesin Superfamily Genes in Cultured Hippocampal Neurons.Cytoskeleton 67: 784-795 Expression of Kinesin Superfamily Genes in Cultured Hippocampal Neurons
kinesin; motor protein; gene expression; neuronal culture; hippocampus
The nature of the different kinesin family members that function in a single, specific neuron type has not been systematically investigated. Here, we used quantitative real-time PCR to analyze the developmental expression patterns of kinesin family genes in cultured mouse hippocampal neurons, a highly homogeneous population of nerve cells. For purposes of comparison, we also determined the set of kinesins expressed in embryonic and adult hippocampal tissue. Twenty kinesins are expressed at moderate-to-high levels in mature hippocampal cultures. These include 9 plus-end directed kinesins from the Kinesin-1, -2, and -3 families that are known to mediate organelle transport and 6 other members of the Kinesin-3 and -4 families that are candidate organelle motors. Hippocampal cultures express high levels of a Kinesin-13, which regulates microtubule depolymerization, and moderate-to-high levels of Kinesin-9 and -14 family members, whose functions are not understood. Twelve additional kinesins, including 10 known mitotic kinesins, are expressed at moderate levels in embryonic hippocampus but at very low levels in mature cultures and the adult hippocampus. Collectively, our findings suggest that kinesins subserve diverse functions within a single type of neuron. (C) 2010 Wiley-Liss, Inc DOI
17. Kwinter, DM; Lo, K; Mafi, P; Silverman, MA. (2009) DYNACTIN REGULATES BIDIRECTIONAL TRANSPORT OF DENSE-CORE VESICLES IN THE AXON AND DENDRITES OF CULTURED HIPPOCAMPAL NEURONS.Neuroscience 162: 1001-1010 DYNACTIN REGULATES BIDIRECTIONAL TRANSPORT OF DENSE-CORE VESICLES IN THE AXON AND DENDRITES OF CULTURED HIPPOCAMPAL NEURONS
CYTOPLASMIC DYNEIN; PLASMINOGEN-ACTIVATOR; VESICULAR TRAFFICKING; SYNAPTIC PLASTICITY; SECRETORY GRANULES; MUTANT DYNACTIN; KINESIN-I; MOTOR; DISEASE; MICROTUBULES
A critical aspect of nerve cell function is peptidergic secretion involving the packaging, transport, and processing of a large group of peptide hormones and other signaling molecules, e.g. brain-derived neurotrophic factor (BDNF). Dense-core vesicles (DCVs) are the organelles that transport these molecules to release sites in both the axon and dendrites of pyramidal neurons. DCVs exhibit complex transport behavior, where these organelles move bidirectionally, reverse direction, pause intermittently, and vary in velocities and run lengths. A key objective in the field of organelle transport is to define the molecules that mediate transport. This study investigated the role of dynactin, a putative opposite-polarity motor coordinator, in the microtubule-based transport of DCVs in primary cultured hippocampal neurons. First, by live cell imaging, we showed similar microtubule-based transport of BDNF, neuropeptide Y (NPY), and tissue plasminogen activator (tPA), consistent with the co-packaging of these DCV cargoes. However, we found higher DCV velocities in both the axon and dendrites than those of previous neuronal studies likely due to faster image acquisition times. Then, using well-characterized dynactin disruptors we demonstrate the need for dynactin in bidirectional transport where overexpression of both p50/dynamitin and the first coiled-coil domain of p150(Glued) (CC1) reduces the flux of DCVs in both directions in the axon and dendrites. We also observed that only CC1 reduces axonal and dendritic run lengths. These results suggest different functions for p50 and p150 in the dynactin complex in DCV transport. These findings are significant because they demonstrate that dynactin functions as a motor coordinator for the transport of DCVs in primary cultured rat hippocampal neurons. Crown Copyright (C) 2009 Published by Elsevier Ltd on behalf of IBRO. All rights reserved. DOI
16.Silverman, MA. (2009) Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons.Journal of Visualized ExperimentsLive Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons
Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images. Vital fluorescent probes, particularly green fluorescent protein (GFP) have revolutionized cell biology stemming from the ability to label specific intracellular compartments and cellular structures. For example, the live imaging of GFP (and its spectral variants) chimeras have allowed for a dynamic analysis of the cytoskeleton, organelle transport, and membrane dynamics in a multitude of organisms and cell types [1-3]. Although live imaging has become prevalent, this approach still poses many technical challenges, particularly in primary cultured neurons. One challenge is the expression of GFP-tagged proteins in post-mitotic neurons; the other is the ability to capture fluorescent images while minimizing phototoxicity, photobleaching, and maintaining general cell health. Here we provide a protocol that describes a lipid-based transfection method that yields a relatively low transfection rate (~0.5%), however is ideal for the imaging of fully polarized neurons. A low transfection rate is essential so that single axons and dendrites can be characterized as to their orientation to the cell body to confirm directionality of transport, i.e., anterograde v. retrograde. Our approach to imaging GFP expressing neurons relies on a standard wide-field fluorescent microscope outfitted with a CCD camera, image capture software, and a heated imaging chamber. We have imaged a wide variety of organelles or structures, for example, dense-core vesicles, mitochondria, growth cones, and actin without any special optics or excitation requirements other than a fluorescent light source. Additionally, spectrally-distinct, fluorescently labeled proteins, e.g., GFP and dsRed-tagged proteins, can be visualized near simultaneously to characterize co-transport or other coordinated cellular events. The imaging approach described here is flexible for a variety of imaging applications and can be adopted by a laboratory for relatively little cost provided a microscope is available.Website DOI
15.Silverman, MA; Leroux, MR. (2009) Intraflagellar transport and the generation of dynamic, structurally and functionally diverse cilia.Trends Cell Biol. 19: 306-316 Intraflagellar transport and the generation of dynamic, structurally and functionally diverse cilia
Cilia are organelles that project from most eukaryotic organisms and cell types. Their pervasiveness stems from having remarkably versatile propulsive and sensory functions, which in humans are recognized to have essential roles in physiology and development. Under-appreciated, however, are their diverse ultrastructures and typically bipartite organization consisting of doublet and singlet microtubules. Moreover, the overall shapes of the membrane-ensheathed cilia are varied, as exemplified by differences between hair-like olfactory cilia and rod- or cone-shaped photoreceptor connecting cilia-outer segments. Although cell-specific transcriptional programs are evidently crucial in establishing ciliary morphological specialization, few players directly involved in generating such diversity are known. Recent findings suggest that at least two molecular motors (kinesin-II and OSM-3/KIF17) can differentially mobilize the intraflagellar transport machinery required for ciliogenesis and, presumably, different cargo to help generate dynamic, structurally and functionally distinct cilia. DOI PubMed
14. Link CD, Fonte V, Roberts CM, Hiester B, Silverman MA, Stein GH. (2008) The beta amyloid peptide can act as a modular aggregation domain.Neurobiology of Disease 32: 420-425 The beta amyloid peptide can act as a modular aggregation domain
Although there is compelling evidence that the beta amyloid peptide (A beta) can be centrally involved in Alzheimer's disease, the natural role (if any) of this peptide remains unclear. Here we use green fluorescent protein (GFP) fusions to demonstrate that the A beta sequence, like prion domains, can act as a modular aggregation domain when terminally appended to a normally soluble protein. We find that a single amino acid substitution (Leu(17) to Pro) in the beta peptide sequence can abolish this cis capacity to induce aggregation. Introduction of this substitution into full-length APP (i.e., a Leu(613)Pro substitution in APP695) alters the processing of APP leading to the accumulation of the C99 C-terminal fragment (CTF). We suggest that in at least some aggregation disease-related proteins the presence of an aggregation domain is not "accidental", but reflects a selected role of these domains in modulating the trafficking or metabolism of the parental protein.
13. Link, CD; Fonte, V; Hiester, B; Yerg, J; Ferguson, J; Csontos, S; Silverman, MA; Stein, GH. (2006) Conversion of green fluorescent protein into a toxic, aggregation-prone protein by C-terminal addition of a short peptide.J Biol Chem 281: 1808-1816 Conversion of green fluorescent protein into a toxic, aggregation-prone protein by C-terminal addition of a short peptide
A non-natural 16-residue "degron" peptide has been reported to convey proteasome-dependent degradation when fused to proteins expressed in yeast (Gilon, T., Chomsky, O., and Kulka, R. ( 2000) Mol. Cell. Biol. 20, 7214-7219) or when fused to green fluorescent protein (GFP) and expressed in mammalian cells (Bence, N. F., Sampat, R. M., and Kopito, R. R. ( 2001) Science 292, 1552-1555). We find that expression of the GFP::degron in Caenorhabditis elegans muscle or neurons results in the formation of stable perinuclear deposits. Similar perinuclear deposition of GFP::degron was also observed upon transfection of primary rat hippocampal neurons or mouse Neuro2A cells. The generality of this observation was supported by transfection of HEK 293 cells with both GFP::degron and DsRed(monomer)::degron constructs. GFP::degron expressed in C. elegans is less soluble than unmodified GFP and induces the small chaperone protein HSP-16, which co-localizes and co-immunoprecipitates with GFP::degron deposits. Induction of GFP::degron in C. elegans muscle leads to rapid paralysis, demonstrating the in vivo toxicity of this aggregating variant. This paralysis is suppressed by co-expression of HSP-16, which dramatically alters the subcellular distribution of GFP::degron. Our results suggest that in C. elegans, and perhaps in mammalian cells, the degron peptide is not a specific proteasome-targeting signal but acts instead by altering GFP secondary or tertiary structure, resulting in an aggregation-prone form recognized by the chaperone system. This altered form of GFP can form toxic aggregates if its expression level exceeds the capacity of chaperone-based degradation pathways. GFP::degron may serve as an instructive "generic" aggregating control protein for studies of disease-associated aggregating proteins, such as huntingtin, alpha-synuclein, and the beta-amyloid peptide.
12. Lochner, JE; Honigman, LS; Grant, WF; Gessford, SK; Hansen, AB; Silverman, MA; Scalettar, BA. (2006) Activity-dependent release of tissue plasminogen activator from the dendritic spines of hippocampal neurons revealed by live-cell imaging.J Neurobiol 66: 564-577 Activity-dependent release of tissue plasminogen activator from the dendritic spines of hippocampal neurons revealed by live-cell imaging
tPA; dense-core granule; regulated secretion; hippocampus; synaptic plasticity
Tissue plasminogen activator (tPA) has been implicated in a variety of important cellular functions, including learning-related synaptic plasticity and potentiating N-methyl-D-aspartate (NMDA) receptor-dependent signaling. These findings suggest that tPA may localize to, and undergo activity-dependent secretion from, synapses; however, conclusive data supporting these hypotheses have remained elusive. To elucidate these issues, we studied the distribution, dynamics, and depolarization-induced secretion of tPA in hippocampal neurons, using fluorescent chimeras of tPA. We found that tPA resides in dense-core granules (DCGs) that traffic to postsynaptic dendritic spines and that can remain in spines for extended periods. We also found that depolarization induced by high potassium levels elicits a slow, partial exocytotic release of tPA from DCGs in spines that is dependent on extracellular Ca+2 concentrations. This slow, partial release demonstrates that exocytosis occurs via a mechanism, such as fuse-pinch-linger, that allows partial release and reuse of DCG cargo and suggests a mechanism that hippocampal neurons may rely upon to avoid depleting tPA at active synapses. Our results also demonstrate release of tPA at a site that facilitates interaction with NMDA-type glutamate receptors, and they provide direct confirmation of fundamental hypotheses about tPA localization and release that bear on its neuromodulatory functions, for example, in learning and memory. (c) 2006 Wiley Periodicals, Inc. DOI
11. Link CD, Fonte V, Hiester B, Yerg J, Ferguson J, Csontos S, Silverman MA, Stein GH. (2005) Conversion of GFP into a toxic, aggregation-prone protein by C-terminal addition of a short peptide.J Biol Chem Oct 19; [Epub ahead of print] Conversion of GFP into a toxic, aggregation-prone protein by C-terminal addition of a short peptide.
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