Patricia Ferrera

β-Amyloid peptide induces ultrastructural changes in synaptosomes and potentiates mitochondrial dysfunction in the presence of ryanodine

In Alzheimers disease (AD), loss of synapses exceeds neuronal loss and some evidence suggests a role of β‐amyloid protein (Aβ) in synaptic degeneration through a mechanism which may involve intraneuronal Ca2+ dyshomeostasis. Emerging evidence points to the participation of the internal Ca2+ stores in the pathophysiology of neurodegeneration in AD. To test the involvement of intrasynaptic Ca2+ mobilization in Aβ toxicity, we explored the role of ryanodine receptor activation in rat cortical synaptosomes taken as a model system for the central presynapses. Evaluation of synaptosomal mitochondrial redox capacity was assessed by the MTT reduction technique, and ultrastructural changes of synaptosomes after exposure to Aβ and ryanodine were evaluated by electron microscopy. Our results show that Aβ potentiates mitochondrial dysfunction in the presence of ryanodine and induces morphological changes consisting of mitochondrial swelling and intense small synaptic vesicles depletion. These changes were accompanied by a reduction in the content of synaptophysin and actin proteins. The reduction of actin immunoreactivity was reversed in the presence of a wide range caspase inhibitors, suggesting the activation of synaptic apoptotic mechanisms.

Short-Term High-Fat-and-Fructose Feeding Produces Insulin Signaling Alterations Accompanied by Neurite and Synaptic Reduction and Astroglial Activation in the Rat Hippocampus

Chronic consumption of high-fat-and-fructose diets (HFFD) is associated with the development of insulin resistance (InsRes) and obesity. Systemic insulin resistance resulting from long-term HFFD feeding has detrimental consequences on cognitive performance, neurogenesis, and long-term potentiation establishment, accompanied by neuronal alterations in the hippocampus. However, diet-induced hippocampal InsRes has not been reported. Therefore, we investigated whether short-term HFFD feeding produced hippocampal insulin signaling alterations associated with neuronal changes in the hippocampus. Rats were fed with a control diet or an HFFD consisting of 10% lard supplemented chow and 20% high-fructose syrup in the drinking water. Our results show that 7 days of HFFD feeding induce obesity and InsRes, associated with the following alterations in the hippocampus: (1) a decreased insulin signaling; (2) a decreased hippocampal weight; (3) a reduction in dendritic arborization in CA1 and microtubule-associated protein 2 (MAP-2) levels; (4) a decreased dendritic spine number in CA1 and synaptophysin content, along with an increase in tau phosphorylation; and finally, (5) an increase in reactive astrocyte associated with microglial changes. To our knowledge, this is the first report addressing hippocampal insulin signaling, as well as morphologic, structural, and functional modifications due to short-term HFFD feeding in the rat.

Estradiol and progesterone modify microtubule associated protein 2 content in the rat hippocampus.

The molecular mechanisms involved in the regulation of synaptic plasticity and neuroprotection by estradiol (E(2)) and progesterone (P(4)) are unknown. Because these processes involve changes in cytoskeleton organization, we studied the effects of E(2) and P(4) in the expression of two cytoskeletal proteins: microtubule associated protein 2 (MAP2) and tau in the hippocampus and the frontal cortex of ovariectomized adult rats. While tau expression was unaffected by E(2) and P(4), an increase in MAP2 protein content in the hippocampus but not in the cortex was observed after E(2) and P(4) treatments. Interestingly, these steroids did not modify MAP2 mRNA content in the hippocampus. These data suggest that MAP2 is involved in the structural changes induced by E(2) and P(4) in the rat hippocampus, and that MAP2 expression is regulated by these steroid hormones at a postranscriptional level.

Cholesterol Potentiates β-Amyloid-Induced Toxicity in Human Neuroblastoma Cells: Involvement of Oxidative Stress

Alterations in brain cholesterol concentration and metabolism seem to be involved in Alzheimer’s disease (AD). In fact, several experimental studies have reported that modification of cholesterol content can influence the expression of the amyloid precursor protein (APP) and amyloid β peptide (Aβ) production. However, it remains to be determined if changes in neuronal cholesterol content may influence the toxicity of Aβ peptides and the mechanism involved. Aged mice, AD patients and neurons exposed to Aβ, show a significant increase in membrane-associated oxidative stress. Since Aβ is able to promote oxidative stress directly by catalytically producing H2O2 from cholesterol, the present work analyzed the effect of high cholesterol incorporated into human neuroblastoma cells in Aβ-mediated neurotoxicity and the role of reactive oxygen species (ROS) generation. Neuronal viability was studied also in the presence of 24S-hydroxycholesterol, the main cholesterol metabolite in brain, as well as the potential protective role of the lipophilic statin, lovastatin.

Sequential expression of cell-cycle regulators and Alzheimer's disease-related proteins in entorhinal cortex after hippocampal excitotoxic damage

Growing evidence suggests that one of the earliest events in the neuronal degeneration of Alzheimers disease (AD) is aberrant cell‐cycle activation in postmitotic neurons, which may, in fact, be sufficient to initiate the neurodegenerative cascade. In the present study we examined whether cyclins and cyclin‐dependent kinases, molecules normally associated with cell‐cycle control, may be involved in delayed expression of altered Alzheimers proteins in two interconnected areas, the entorhinal cortex (EC) and the dentate gyrus (DG), after a hippocampal excitotoxic lesion. Several cell‐cycle proteins of the G1 and S phases and even of the G2 phase were found to be up‐regulated in the EC after kainic acid evoked neuronal death in the hippocampus. In addition, we describe the progressive expression of two Alzheimers‐related proteins, PHF‐1 and APP, which reached higher levels immediately after the increase in G1/S‐phase markers. Hence, the results of the present study support the participation of cell‐cycle dysregulation as a key component of the process that may ultimately lead to expression of AD proteins and neuronal death in a brain area when the target site for synaptic inputs in that area is damaged by an excitotoxic insult.

Amyloid-β protein modulates insulin signaling in presynaptic terminals.

Synaptic loss is a major neuropathological correlate of memory decline as a result of Alzheimer’s disease (AD). This phenomenon appears to be aggravated by soluble amyloid-β (Aβ) oligomers causing presynaptic terminals to be particularly vulnerable to damage. Furthermore, insulin is known to participate in synaptic plasticity through the activation of the insulin receptor (IR) and the PI3K signaling pathway, while low concentrations of soluble Aβ and Aβ oligomers aberrantly modulate IR function in cultured neurons. To further examine how Aβ and insulin interact in the pathology of AD, the present work analyzes the effect of insulin and Aβ in the activation of the IR/PI3K pathway in synaptosomes. We found that insulin increased mitochondrial activity and IR/Akt phosphorylation in synaptosomes taken from both hippocampus and cortex. Also, pretreatment with Aβ antagonized insulin’s effect on hippocampal synaptosomes, but not vice versa. These results show that Aβ can reduce responsiveness to insulin. Combined with evidence that insulin desensitization can increase the risk of developing AD, our results suggest that the initial mechanism that impairs synaptic maintenance in AD might start with Aβ changes in insulin sensitivity.

Caspase-12 activation is involved in amyloid-β protein-induced synaptic toxicity.

Synapse loss is considered to be the best correlate of cognitive impairments in Alzheimers disease (AD), and growing evidence supports the notion that certain events that trigger neuronal death in AD can be initiated by the local activation of caspases within the synaptic compartment. We have demonstrated previously that presynaptic terminals are particularly vulnerable to endoplasmic-reticulum (ER)-stress depending of amyloid-β protein (Aβ). This toxicity included a notable reduction of actin and synaptophysin protein and mitochondrial dysfunction. This synaptic damage was prevented by incubation with a wide range of caspase inhibitor, suggesting the activation of local synaptic apoptotic mechanisms. The ER-resident caspase-12 was initially identified as a mediator of Aβ neurotoxicity. Thus, the current study was conducted to explore the presence and local activation of the caspase-12 in cortical and hippocampal synaptosomes isolated from rat and from the triple transgenic mouse model of AD (3xTg-AD) in the presence of Aβ and ryanodine. Under these conditions, we found mitochondrial failure accompanied by a reduction in actin levels which was dependent on caspase-12 activation suggesting its participation in Aβ-induced synaptic toxicity.

Differential effects of COX inhibitors against β-amyloid-induced neurotoxicity in human neuroblastoma cells

Retrospective epidemiological studies have suggested that chronic treatment with nonsteroidal anti-inflammatory drugs (NSAIDs) provides some degree of protection from Alzheimers disease (AD). Although most NSAIDs inhibit the activity of cyclooxygenase (COX), the rate-limiting enzyme in the production of prostanoids from arachidonic acid (AA), the precise mechanism through which NSAIDs act upon AD pathology remains to be elucidated. Classical NSAIDs like indomethacin inhibit both the constitutive COX-1 and the inducible COX-2 enzymes. In the present work, we characterize the protective effect of the indomethacin on the neurotoxicity elicited by amyloid-beta protein (A beta, fragments 25-35 and 1-42) alone or in combination with AA added exogenously as well as its effects on COX-2 expression. We also compared the neuroprotective effects of indomethacin with the selective COX-1, COX-2 and 5-LOX inhibitors, SC-560, NS-398 and NDGA, respectively. Our results show that indomethacin protected from A beta and AA toxicity in naive and differentiated human neuroblastoma cells with more potency than SC-560 while, NS-398 only protected neurons from AA-mediated toxicity. Present results suggest that A beta toxicity can be reversed more efficiently by the non-selective COX inhibitor indomethacin suggesting its role in modulating the signal transduction pathway involved in the mechanism of A beta neurotoxicity.

Okadaic acid induces epileptic seizures and hyperphosphorylation of the NR2B subunit of the NMDA receptor in rat hippocampus in vivo

Overactivation of N-methyl-D-aspartate (NMDA) glutamate receptors is closely related to epilepsy and excitotoxicity, and the phosphorylation of these receptors may facilitate glutamate-mediated synaptic transmission. Here we show that in awake rats the microinjection into the hippocampus of okadaic acid, a potent inhibitor of protein phosphatases 1 and 2A, induces in about 20 min intense electroencephalographic and behavioral limbic-type seizures, which are suppressed by the systemic administration of the NMDA receptor antagonist (+)-5-methyl-10,11-dihydro-5H-dibenzo-[a,d]cyclohepten-5,10-imine hydrogen maleate and by the intrahippocampal administration of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, an inhibitor of protein kinases. Two hours after okadaic acid, when the EEG seizures were intense, an increased serine phosphorylation of some hippocampal proteins, including an enhancement of the serine phosphorylation of the NMDA receptor subunit NR2B, was detected by immunoblotting. Twenty-four hours after okadaic acid a marked destruction of hippocampal CA1 region was observed, which was not prevented by the receptor antagonists. These findings suggest that hyperphosphorylation of glutamate receptors in vivo may result in an increased sensitivity to the endogenous transmitter and therefore induce neuronal hyperexcitability and epilepsy.

Interplay between cholesterol and homocysteine in the exacerbation of amyloid-β toxicity in human neuroblastoma cells.

Amyloid-β (Aβ) plays an important role in Alzheimers disease (AD) progression and is associated with synaptic damage and neuronal death. Epidemiological and experimental studies indicate that hypercholesterolemia and hyperhomocysteinemia increase susceptibility to AD; however, the exact impact and mechanisms involved are largely unknown. Few studies have addressed the combined effects of the above compounds, which are considered to be risk factors for developing AD, on Aβ-induced neurotoxicity. The aim of the present work was to analyze the relationships between homocysteine (Hcy) and cholesterol and their role in Aβ toxicity in human neuroblastoma cells, as well as the mechanisms associated with this neurotoxicity. In addition to finding that Hcy is involved in cholesterol homeostasis in neurons, we demonstrate that the combined effect of cholesterol and Hcy in the presence of copper significantly increases the levels of reactive oxygen species and may render neurons more vulnerable to Aβ.