Islet amyloid polypeptide (IAPP, amylin) is the major protein component of the islet amyloid deposits associated with type 2 diabetes. The polypeptide lacks a well-defined structure in its monomeric state but readily assembles to form amyloid. Amyloid fibrils formed from TAPP, intermediates generated in the assembly of IAPP amyloid, or both are toxic to beta-cells, suggesting that islet amyloid formation may contribute to the pathology of type 2 diabetes. There are relatively few reported inhibitors of amyloid formation by IAPP. Here we show that the tea-derived flavanol, (-)-epigallocatechin 3-gallate [(2R,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)-3,4-dihydro-2H-1-benzopyran-3-yl 3,4,5-trihydroxybenzoate] (EGCG), is an effective inhibitor of in vitro IAPP amyloid formation and disaggregates preformed amyloid fibrils derived from IAPP. The compound is thus one of a very small set of molecules which have been shown to disaggregate TAPP amyloid fibrils. Fluorescence-detected thioflavin-T binding assays and transmission electron microscopy confirm that the compound inhibits unseeded amyloid fibril formation as well as disaggregates IAPP amyloid. Seeding studies show that the complex formed by IAPP and EGCG does not seed amyloid formation by LAPP. In this regard, the behavior of IAPP is similar to the reported interactions of A beta and alpha-synuclein with EGCG. Alamar blue assays and light microscopy indicate that the compound protects cultured rat INS-1 cells against IAPP-induced toxicity. Thus, EGCG offers an interesting lead structure for further development of inhibitors of IAPP amyloid formation and compounds that disaggregate IAPP amyloid.
It has been suggested that accumulation of beta‐amyloid (Aβ) peptide triggers neurodegeneration, at least in part, via glutamate‐mediated excitotoxicity in Alzheimer’s disease (AD) brain. This is supported by observations that toxicity induced by Aβ peptide in cultured neurons and in adult rat brain is known to be mediated by activation of glutamatergic N‐methyl‐d‐aspartate (NMDA) receptors. Additionally, recent clinical studies have shown that memantine, a noncompetitive NMDA receptor antagonist, can significantly improve cognitive functions in some AD patients. However, very little is currently known about the potential role of memantine against Aβ‐induced toxicity. In the present study, we have shown that Aβ1–42‐induced toxicity in rat primary cortical cultured neurons is accompanied by increased extracellular and decreased intracellular glutamate levels. We subsequently demonstrated that Aβ toxicity is induced by increased phosphorylation of tau protein and activation of tau kinases, i.e. glycogen synthase kinase‐3β and extracellular signal‐related kinase 1/2. Additionally, Aβ treatment induced cleavage of caspase‐3 and decreased phosphorylation of cyclic AMP response element binding protein, which are critical in determining survival of neurons. Memantine treatment significantly protected cultured neurons against Aβ‐induced toxicity by attenuating tau‐phosphorylation and its associated signaling mechanisms. However, this drug did not alter either conformation or internalization of Aβ1–42 and it was unable to attenuate Aβ‐induced potentiation of extracellular glutamate levels. These results, taken together, provide new insights into the possible neuroprotective action of memantine in AD pathology.
2-Deoxy-d-[14C]glucose ([14C]DG) is commonly used to determine local glucose utilization rates (CMRglc) in living brain and to estimate CMRglc in cultured brain cells as rates of [14C]DG phosphorylation. Phosphorylation rates of [14C]DG and its metabolizable fluorescent analog, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG), however, do not take into account differences in the kinetics of transport and metabolism of [14C]DG or 2-NBDG and glucose in neuronal and astrocytic cells in cultures or in single cells in brain tissue, and conclusions drawn from these data may, therefore, not be correct. As a first step toward the goal of quantitative determination of CMRglc in astrocytes and neurons in cultures, the steady-state intracellular-to-extracellular concentration ratios (distribution spaces) for glucose and [14C]DG were determined in cultured striatal neurons and astrocytes as functions of extracellular glucose concentration. Unexpectedly, the glucose distribution spaces rose during extreme hypoglycemia, exceeding 1.0 in astrocytes, whereas the [14C]DG distribution space fell at the lowest glucose levels. Calculated CMRglc was greatly overestimated in hypoglycemic and normoglycemic cells because the intracellular glucose concentrations were too high. Determination of the distribution space for [14C]glucose revealed compartmentation of intracellular glucose in astrocytes, and probably, also in neurons. A smaller metabolic pool is readily accessible to hexokinase and communicates with extracellular glucose, whereas the larger pool is sequestered from hexokinase activity. A new experimental approach using double-labeled assays with DG and glucose is suggested to avoid the limitations imposed by glucose compartmentation on metabolic assays.
Autophagic (type II) cell death, characterized by the massive accumulation of autophagic vacuoles in the cytoplasm of cells, has been suggested to play pathogenetic roles in cerebral ischemia, brain trauma, and neurodegenerative disorders. 3,4-Methylenedioxymethamphetamine (MDMA or ecstasy) is an illicit drug causing long-term neurotoxicity in the brain. Apoptotic (type I) and necrotic (type III) cell death have been implicated in MDMA-induced neurotoxicity, while the role of autophagy in MDMA-elicited neurotoxicity has not been investigated. The present study aimed to evaluate the occurrence and contribution of autophagy to neurotoxicity in cultured rat cortical neurons challenged with MDMA. Autophagy activation was monitored by expression of microtubule-associated protein 1 light chain 3 (LC3; an autophagic marker) using immunofluorescence and western blot analysis. Here, we demonstrate that MDMA exposure induced monodansylcadaverine (MDC)- and LC3B-densely stained autophagosome formation and increased conversion of LC3B-I to LC3B-II, coinciding with the neurodegenerative phase of MDMA challenge. Autophagy inhibitor 3-methyladenine (3-MA) pretreatment significantly attenuated MDMA-induced autophagosome accumulation, LC3B-II expression, and ameliorated MDMA-triggered neurite damage and neuronal death. In contrast, enhanced autophagy flux by rapamycin or impaired autophagosome clearance by bafilomycin A1 led to more autophagosome accumulation in neurons and aggravated neurite degeneration, indicating that excessive autophagosome accumulation contributes to MDMA-induced neurotoxicity. Furthermore, MDMA induced phosphorylation of AMP-activated protein kinase (AMPK) and its downstream unc-51-like kinase 1 (ULK1), suggesting the AMPK/ULK1 signaling pathway might be involved in MDMA-induced autophagy activation.
Type 2 diabetes mellitus is a metabolic disorder characterized by hyperglycemia and is especially prevalent in the elderly. Because aging is a risk factor for type 2 diabetes mellitus, and insulin resistance may contribute to the pathogenesis of Alzheimer’s disease (AD), anti‐diabetic agents (thiazolidinediones‐TZDs) are being studied for the treatment of cognitive decline associated with AD. These agents normalize insulin sensitivity in the periphery and can improve cognition and verbal memory in AD patients. Based on evidence that Ca2+ dysregulation is a pathogenic factor of brain aging/AD, we tested the hypothesis that TZDs could impact Ca2+ signaling/homeostasis in neurons. We assessed the effects of pioglitazone and rosiglitazone (TZDs) on two major sources of Ca2+ influx in primary hippocampal cultured neurons, voltage‐gated Ca2+ channel (VGCC) and the NMDA receptor (NMDAR). VGCC‐ and NMDAR‐mediated Ca2+ currents were recorded using patch‐clamp techniques, and Ca2+ intracellular levels were monitored with Ca2+ imaging techniques. Rosiglitazone, but not pioglitazone reduced VGCC currents. In contrast, NMDAR‐mediated currents were significantly reduced by pioglitazone but not rosiglitazone. These results show that TZDs modulate Ca2+‐dependent pathways in the brain and have different inhibitory profiles on two major Ca2+ sources, potentially conferring neuroprotection to an area of the brain that is particularly vulnerable to the effects of aging and/or AD.
Post-mortem analyses of brains from patients with Parkinson disease who received fetal mesencephalic transplants show that alpha-synuclein-containing (alpha-syn-containing) Lewy bodies gradually appear in grafted neurons. Here, we explored whether intercellular transfer of alpha-syn from host to graft, followed by seeding of alpha-syn aggregation in recipient neurons, can contribute to this phenomenon. We assessed alpha-syn cell-to-cell transfer using microscopy, flow cytometry, and high-content screening in several coculture model systems. Coculturing cells engineered to express either GFP- or DsRed-tagged alpha-syn resulted in a gradual increase in double-labeled cells. Importantly, alpha-syn-GFP derived from 1 neuroblastoma cell line localized to red fluorescent aggregates in other cells expressing DsRed-alpha-syn, suggesting a seeding effect of transmitted alpha-syn. Extracellular alpha-syn was taken up by cells through endocytosis and interacted with intracellular alpha-syn. Next, following intracortical injection of recombinant alpha-syn in rats, we found neuronal uptake was attenuated by coinjection of an endocytosis inhibitor. Finally, we demonstrated in vivo transfer of alpha-syn between host cells and grafted dopaminergic neurons in mice overexpressing human alpha-syn. In summary, intercellularly transferred alpha-syn interacts with cytoplasmic alpha-syn and can propagate alpha-syn pathology. These results suggest that alpha-syn propagation is a key element in the progression of Parkinson disease pathology.
Accumulating evidence shows that several cell types have the capacity to secrete membrane proteins by incorporating them into exosomes, which are small lipid vesicles derived from the intralumenal membranes of multivesicular bodies (MVBs) of the endocytic pathway. Exosomes are expelled in the extracellular space upon fusion of the MVB with the plasma membrane. Exosomal release is a way of secreting membrane proteins meant to be discarded, or to be passed on to other cells. Here, we demonstrate, using primary cortical cultures, that neurones and astrocytes can secrete exosomes. We find that exosomes released by cortical neurones contain the L1 cell adhesion molecule, the GPI-anchored prion protein, and the GluR2/3 but not the NR1 subunits of glutamate receptors. We also show that exosomal release is regulated by depolarisation. Our observation suggests that exosomes may have a regulatory function at synapses and could also allow intercellular exchange of membrane proteins within the brain.
The morphology of astrocytes, likely regulated by cAMP, determines the structural association between astrocytes and the synapse, consequently modulating synaptic function. β‐Adrenergic receptors (β‐AR), which increase cytosolic cAMP concentration ([cAMP]i), may affect cell morphology. However, the real‐time dynamics of β‐AR‐mediated cAMP signaling in single live astrocytes and its effect on cell morphology have not been studied. We used the fluorescence resonance energy transfer (FRET)‐based cAMP biosensor Epac1‐camps to study time‐dependent changes in [cAMP]i; morphological changes in primary rat astrocytes were monitored by real‐time confocal microscopy. Stimulation of β‐AR by adrenaline, noradrenaline, and isoprenaline, a specific agonist of β‐AR, rapidly increased [cAMP]i (∼15 s). The FRET signal response, mediated via β‐AR, was faster than in the presence of forskolin (twofold) and dibutyryl‐cAMP (>35‐fold), which directly activate adenylyl cyclase and Epac1‐camps, respectively, likely due to slow entry of these agents into the cytosol. Oscillations in [cAMP]i have not been recorded, indicating that cAMP‐dependent processes operate in a slow time domain. Most Epac1‐camps expressing astrocytes revealed a morphological change upon β‐AR activation and attained a stellate morphology within 1 h. The morphological changes exhibited a bell‐shaped dependency on [cAMP]i. The 5–10% decrease in cell cross‐sectional area and the 30–50% increase in cell perimeter are likely due to withdrawal of the cytoplasm to the perinuclear region and the appearance of protrusions on the surface of astrocytes. Because astrocyte processes ensheath neurons, β‐AR/cAMP‐mediated morphological changes can modify the geometry of the extracellular space, affecting synaptic, neuronal, and astrocyte functions in health and disease. GLIA 2014;62:566–579 Main Points The results show the first β‐Adrenergic receptor evoked time‐dependent changes in cytosolic cAMP in astrocytes measured by FRET nanosensor and subsequent changes in astrocyte morphology that exhibited bell‐shaped dependency on cytosolic cAMP levels.
Our previous studies reported that methanol extract of Sanguisorbae radix from Sanguisorba officinalis L. (Rosaceae) prevented neuronal cell damage induced by Aβ (25-35) in vitro. The present study was carried out to investigate the effect of gallic acid isolated from Sanguisorbae radix on Aβ (25-35)-induced neurotoxicity using cultured rat cortical neurons. Gallic acid (0.1, 1 μM) showed a concentration-dependent inhibition on Aβ (25-35) (10 μM)-induced apoptotic neuronal death, as assessed by a 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay and Hoechst 33342 staining. Pretreatment of gallic acid inhibited 10 μM Aβ (25-35)-induced elevation of cytosolic Ca2+ concentration ([Ca2+]c) and generation of reactive oxygen species (ROS), which were measured by fluorescent dyes. Gallic acid also inhibited glutamate release into medium induced by 10 μM Aβ (25-35), which was measured by HPLC. These results suggest that gallic acid prevents Aβ (25-35)-induced apoptotic neuronal death by interfering with the increase of [Ca2+]c, and then by inhibiting glutamate release and generation of ROS, and that these effects of gallic acid may be partly associated with the neuroprotective effect of Sanguisorbae radix.