The deposition of amyloid‐β (Aβ) peptide, which is generated from amyloid precursor protein (APP), is the pathological hallmark of Alzheimer's disease (AD). Three APP familial AD mutations (D678H, D678N, and H677R) located at the sixth and seventh amino acid of Aβ have distinct effect on Aβ aggregation, but their influence on the physiological and pathological roles of APP remain unclear. We found that the D678H mutation strongly enhances amyloidogenic cleavage of APP, thus increasing the production of Aβ. This enhancement of amyloidogenic cleavage is likely because of the acceleration of APPD678H sorting into the endosomal‐lysosomal pathway. In contrast, the APPD678N and APPH677R mutants do not cause the same effects. Therefore, this study indicates a regulatory role of D678H in APP sorting and processing, and provides genetic evidence for the importance of APP sorting in AD pathogenesis.
Expression of a familial Alzheimer's disease (AD)‐linked mutant of amyloid β precursor protein (APP) or the binding of transforming growth factor β2 to wild‐type (wt)‐APP causes neuronal death by activating an intracellular death signal (a APP‐mediated intracellular death signal) in the absence of the involvement of amyloid β (Aβ) toxicity in vitro. These neuronal death models may therefore be regarded as Aβ‐independent neuronal death models related to AD. A recent study has shown that the A673T mutation in the APP isoform APP770, corresponding to the A598T mutation in the most prevalent neuronal APP isoform APP695 (an AD‐protective mutant of APP), is linked to a reduction in the incidence rate of AD. Consistent with this, cells expressing the AD‐protective mutant of APP produce less Aβ than cells expressing wt‐APP. In this study, transforming growth factor β2 caused death in cultured neuronal cells expressing wt‐APP, but not in those expressing the AD‐protective mutant of APP. This result suggests that the AD‐protective mutation of APP reduces the incidence rate of AD by attenuating the APP‐mediated intracellular death signal. In addition, a mutation that causes hereditary cerebral hemorrhage with amyloidosis‐Dutch type also attenuated the APP‐mediated intracellular death signal.
Cholinergic signaling plays an important role in regulating the growth and regeneration of axons in the nervous system. The α7 nicotinic receptor (α7) can drive synaptic development and plasticity in the hippocampus. Here, we show that activation of α7 significantly reduces axon growth in hippocampal neurons by coupling to G protein‐regulated inducer of neurite outgrowth 1 (Gprin1), which targets it to the growth cone. Knockdown of Gprin1 expression using RNAi is found sufficient to abolish the localization and calcium signaling of α7 at the growth cone. In addition, an α7/Gprin1 interaction appears intimately linked to a Gαo, growth‐associated protein 43, and CDC42 cytoskeletal regulatory pathway within the developing axon. These findings demonstrate that α7 regulates axon growth in hippocampal neurons, thereby likely contributing to synaptic formation in the developing brain.
Methylone, 3,4‐methylenedioxypyrovalerone (MDPV), and mephedrone are psychoactive ingredients of ‘bath salts’ and their abuse represents a growing public health care concern. These drugs are cathinone derivatives and are classified chemically as β‐ketoamphetamines. Because of their close structural similarity to the amphetamines, methylone, MDPV, and mephedrone share most of their pharmacological, neurochemical, and behavioral properties. One point of divergence in their actions is the ability to cause damage to the CNS. Unlike methamphetamine, the β‐ketoamphetamines do not damage dopamine (DA) nerve endings. However, mephedrone has been shown to significantly accentuate methamphetamine neurotoxicity. Bath salt formulations contain numerous different psychoactive ingredients, and individuals who abuse bath salts also coabuse other illicit drugs. Therefore, we have evaluated the effects of methylone, MDPV, mephedrone, and methamphetamine on DA nerve endings. The β‐ketoamphetamines alone or in all possible two‐drug combinations do not result in damage to DA nerve endings but do cause hyperthermia. MDPV completely protects against the neurotoxic effects of methamphetamine while methylone accentuates it. Neither MDPV nor methylone attenuates the hyperthermic effects of methamphetamine. The potent neuroprotective effects of MDPV extend to amphetamine‐, 3,4‐methylenedioxymethamphetamine‐, and MPTP‐induced neurotoxicity. These results indicate that β‐ketoamphetamine drugs that are non‐substrate blockers of the DA transporter (i.e., MDPV) protect against methamphetamine neurotoxicity, whereas those that are substrates for uptake by the DA transporter and which cause DA release (i.e., methylone, mephedrone) accentuate neurotoxicity.
The receptor for advanced glycation end products (RAGE) gene expresses two major alternative splicing isoforms, full‐length membrane‐bound RAGE (mRAGE) and secretory RAGE (esRAGE). Both isoforms play important roles in Alzheimer's disease (AD) pathogenesis, either via interaction of mRAGE with β‐amyloid peptide (Aβ) or inhibition of the mRAGE‐activated signaling pathway. In the present study, we showed that heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) and Transformer2β‐1 (Tra2β‐1) were involved in the alternative splicing of mRAGE and esRAGE. Functionally, two factors had an antagonistic effect on the regulation. Glucose deprivation induced an increased ratio of mRAGE/esRAGE via up‐regulation of hnRNP A1 and down‐regulation of Tra2β‐1. Moreover, the ratios of mRAGE/esRAGE and hnRNP A1/Tra2β‐1 were increased in peripheral blood mononuclear cells from AD patients. The results provide a molecular basis for altered splicing of mRAGE and esRAGE in AD pathogenesis.
Leptin is a centrally acting hormone that controls metabolic pathways. Recent epidemiological studies suggest that plasma leptin is protective against Alzheimer's disease. However, the mechanism that underlies this effect remains uncertain. To investigate whether leptin inhibits the assembly of amyloid β‐protein (Aβ) on the cell surface of neurons, we treated primary neurons with leptin. Leptin treatment decreased the GM1 ganglioside (GM1) levels in the detergent‐resistant membrane microdomains (DRMs) of neurons. The increase in GM1 expression induced by leptin was inhibited after pre‐treatment with inhibitors of phosphatidylinositol 3‐kinase (LY294002), Akt (triciribine) and the mammalian target of rapamycin (i.e. rapamycin), but not by an inhibitor of extracellular signal‐regulated kinase (PD98059). In addition, pre‐treatment with these reagents blocked the induction of GM1 in DRMs by leptin. Furthermore, Aβ assembly on the cell surface of neurons was inhibited greatly after treatment with leptin. This reduction was markedly inhibited after pre‐treatment with LY294002, triciribine, and rapamycin. These results suggest that leptin significantly inhibits Aβ assembly by decreasing GM1 expression in DRMs of the neuronal surface through the phosphatidylinositol 3‐kinase/Akt/mammalian target of rapamycin pathway.
Diet supplementation with ketone bodies (acetoacetate and β‐hydroxybuturate) or medium‐length fatty acids generating ketone bodies has consistently been found to cause modest improvement of mental function in Alzheimer's patients. It was suggested that the therapeutic effect might be more pronounced if treatment was begun at a pre‐clinical stage of the disease instead of well after its manifestation. The pre‐clinical stage is characterized by decade‐long glucose hypometabolism in brain, but ketone body metabolism is intact even initially after disease manifestation. One reason for the impaired glucose metabolism may be early destruction of the noradrenergic brain stem nucleus, locus coeruleus, which stimulates glucose metabolism, at least in astrocytes. These glial cells are essential in Alzheimer pathogenesis. The β‐amyloid peptide Aβ interferes with their cholinergic innervation, which impairs synaptic function because of diminished astrocytic glutamate release. Aβ also reduces glucose metabolism and causes hyperexcitability. Ketone bodies are similarly used against seizures, but the effectively used concentrations are so high that they must interfere with glucose metabolism and de novo synthesis of neurotransmitter glutamate, reducing neuronal glutamatergic signaling. The lower ketone body concentrations used in Alzheimer's disease may owe their effect to support of energy metabolism, but might also inhibit release of gliotransmitter glutamate.
The amyloid precursor protein (APP) and its mammalian homologs, APLP1, APLP2, have been allocated to an organellar pool residing in the Golgi apparatus and in endosomal compartments, and in its mature form to a cell surface‐localized pool. In the brain, all APPs are restricted to neurons; however, their precise localization at the plasma membrane remained enigmatic. Employing a variety of subcellular fractionation steps, we isolated two synaptic vesicle (SV) pools from rat and mouse brain, a pool consisting of synaptic vesicles only and a pool comprising SV docked to the presynaptic plasma membrane. Immunopurification of these two pools using a monoclonal antibody directed against the 12 membrane span synaptic vesicle protein2 (SV2) demonstrated unambiguously that APP, APLP1 and APLP2 are constituents of the active zone of murine brain but essentially absent from free synaptic vesicles. The specificity of immunodetection was confirmed by analyzing the respective knock‐out animals. The fractionation experiments further revealed that APP is accumulated in the fraction containing docked synaptic vesicles. These data present novel insights into the subsynaptic localization of APPs and are a prerequisite for unraveling the physiological role of all mature APP proteins in synaptic physiology.
Glucose is the main energy substrate for neurons, and ketone bodies are known to be alternative substrates. However, the capacity of ketone bodies to support different neuronal functions is still unknown. Thus, a change in energy substrate from glucose alone to a combination of glucose and β‐hydroxybutyrate might change neuronal function as there is a known coupling between metabolism and neurotransmission. The purpose of this study was to shed light on the effects of the ketone body β‐hydroxybutyrate on glycolysis and neurotransmission in cultured murine glutamatergic neurons. Previous studies have shown an effect of β‐hydroxybutyrate on glucose metabolism, and the present study further specified this by showing attenuation of glycolysis when β‐hydroxybutyrate was present in these neurons. In addition, the NMDA receptor‐induced calcium responses in the neurons were diminished in the presence of β‐hydroxybutyrate, whereas a direct effect of the ketone body on transmitter release was absent. However, the presence of β‐hydroxybutyrate augmented transmitter release induced by the KATP channel blocker glibenclamide, thus giving an indirect indication of the involvement of KATP channels in the effects of ketone bodies on transmitter release.
Chronic neuropathic pain is a common consequence of spinal cord injury (SCI), develops over time and negatively impacts quality of life, often leading to substance abuse and suicide. Recent evidence has demonstrated that reactive oxygen species (ROS) play a role in contributing to neuropathic pain in SCI animal models. This investigation examines four compounds that reduce ROS and the downstream lipid peroxidation products, apocynin, 4‐oxo‐tempo, U‐83836E, and tirilazad, and tests if these compounds can reduce nocioceptive behaviors in chronic SCI animals. Apocynin and 4‐oxo‐tempo significantly reduced abnormal mechanical hypersensitivity measured in forelimbs and hindlimbs in a model of chronic SCI‐induced neuropathic pain. Thus, compounds that inhibit ROS or lipid peroxidation products can be used to ameliorate chronic neuropathic pain.
Neuronal cells are characterized by the presence of two confined domains, which are different in their cellular properties, biochemical functions and molecular identity. The generation of asymmetric domains in neurons should logically require specialized membrane trafficking to both promote neurite outgrowth and differential distribution of components. Members of the Rab family of small GTPases are key regulators of membrane trafficking involved in transport, tethering and docking of vesicles through their effectors. RabGTPases activity is coupled to the activity of guanine nucleotide exchange factors or GEFs, and GTPase‐activating proteins known as GAPs. Since the overall spatiotemporal distribution of GEFs, GAPs and Rabs governs trafficking through the secretory and endocytic pathways, affecting exocytosis, endocytosis and endosome recycling, it is likely that RabGTPases could have a major role in neurite outgrowth, elongation and polarization. In this review we summarize the evidence linking the functions of several RabGTPases to axonal and dendritic development in primary neurons, as well as neurite formation in neuronal cell lines. We focused on the role of RabGTPases from the trans‐Golgi network, early/late and recycling endosomes, as well as the function of some Rab effectors in neuritogenesis. Finally, we also discuss the participation of the ADP‐ribosylation factor 6, a member of the ArfGTPase family, in neurite formation since it seems to have an important cross‐talk with RabGTPases.
The 19‐transmembrane, multisubunit γ‐secretase complex generates the amyloid β‐peptide (Aβ) of Alzheimer's disease (AD) by an unusual intramembrane proteolysis of the β‐amyloid precursor protein. The complex, which similarly processes many other type 1 transmembrane substrates, is composed of presenilin, Aph1, nicastrin, and presenilin enhancer (Pen‐2), all of which are necessary for proper complex maturation and enzymatic activity. Obtaining a high‐resolution atomic structure of the intact complex would greatly aid the rational design of compounds to modulate activity but is a very difficult task. A complementary method is to generate structures for each individual subunit to allow one to build a model of the entire complex. Here, we describe a method by which recombinant human Pen‐2 can be purified from bacteria to > 95% purity at milligram quantities per liter, utilizing a maltose binding protein tag to both increase solubility and facilitate purification. Expressing the same construct in mammalian cells, we show that the large N‐terminal maltose binding protein tag on Pen‐2 still permits incorporation into the complex and subsequent presenilin‐1 endoproteolysis, nicastrin glycosylation and proteolytic activity. These new methods provide valuable tools to study the structure and function of Pen‐2 and the γ‐secretase complex.
The function of amyloid precursor protein (APP) is unknown, although the discovery that it contributes to the regulation of surface expression of N‐methyl‐d ‐aspartate (NMDA) receptors has afforded new insights into its functional significance. Since APP is a member of a gene family that contains two other members, amyloid precursor‐like proteins 1 and 2 (APLP1 and APLP2), it is important to determine if the related APP proteins possess the same properties as APP with respect to their interactions with NMDA receptors. Following expression in mammalian cells, both APLP1 and APLP2 behaved similarly to APP in that they both co‐immunoprecipitated with the two major NMDA receptor subtypes, GluN1/GluN2A and GluN1/GluN2B, via interaction with the obligatory GluN1 subunit. Immunoprecipitations from detergent extracts of adult mammalian brain showed co‐immunoprecipitation of APLP1 and APLP2 with GluN2A‐ and GluN2B‐containing NMDA receptors. Furthermore, similarly to APP, APLP1 and APLP2 both enhanced GluN1/GluN2A and GluN1/GluN2B cell surface expression. Thus, all the three members of the APP gene family behave similarly in that they each contribute to the regulation of cell surface NMDA receptor homoeostasis.
Glutamate transport is a critical process in the brain that maintains low extracellular levels of glutamate to allow for efficient neurotransmission and prevent excitotoxicity. Loss of glutamate transport function is implicated in epilepsy, traumatic brain injury, and amyotrophic lateral sclerosis. It remains unclear whether or not glutamate transport can be modulated in these disease conditions to improve outcome. Here, we show that sirtuin (SIRT)4, a mitochondrial sirtuin, is up‐regulated in response to treatment with the potent excitotoxin kainic acid. Loss of SIRT4 leads to a more severe reaction to kainic acid and decreased glutamate transporter expression and function in the brain. Together, these results indicate a critical and novel stress response role for SIRT4 in promoting proper glutamate transport capacity and protecting against excitotoxicity.
Localized translation of axonal mRNAs contributes to developmental and regenerative axon growth. Although untranslated regions (UTRs) of many different axonal mRNAs appear to drive their localization, there has been no consensus RNA structure responsible for this localization. We recently showed that limited expression of ZBP1 protein restricts axonal localization of both β‐actin and GAP‐43 mRNAs. β‐actin 3′UTR has a defined element for interaction with ZBP1, but GAP‐43 mRNA shows no homology to this RNA sequence. Here, we show that an AU‐rich regulatory element (ARE) in GAP‐43′s 3′UTR is necessary and sufficient for its axonal localization. Axonal GAP‐43 mRNA levels increase after in vivo injury, and GAP‐43 mRNA shows an increased half‐life in regenerating axons. GAP‐43 mRNA interacts with both HuD and ZBP1, and HuD and ZBP1 co‐immunoprecipitate in an RNA‐dependent fashion. Reporter mRNA with the GAP‐43 ARE competes with endogenous β‐actin mRNA for axonal localization and decreases axon length and branching similar to the β‐actin 3′UTR competing with endogenous GAP‐43 mRNA. Conversely, over‐expressing GAP‐43 coding sequence with its 3′UTR ARE increases axonal elongation and this effect is lost when just the ARE is deleted from GAP‐43′s 3′UTR.
Huntington's disease (HD) is one of many neurodegenerative diseases with reported alterations in brain iron homeostasis that may contribute to neuropathogenesis. Iron accumulation in the specific brain areas of neurodegeneration in HD has been proposed based on observations in post‐mortem tissue and magnetic resonance imaging studies. Altered magnetic resonance imaging signal within specific brain regions undergoing neurodegeneration has been consistently reported and interpreted as altered levels of brain iron. Biochemical studies using various techniques to measure iron species in human samples, mouse tissue, or in vitro has generated equivocal data to support such an association. Whether elevated brain iron occurs in HD, plays a significant contributing role in HD pathogenesis, or is a secondary effect remains currently unclear.
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