Hexa-Histidine, a Peptide with Versatile Applications in the Study of Amyloid-ß(1-42) Molecular Mechanisms of Action.

Amyloid ß (Aß) oligomers are the most neurotoxic forms of Aß, and Aß(1-42) is the prevalent Aß peptide found in the amyloid plaques of Alzheimer's disease patients. Aß(25-35) is the shortest peptide that retains the toxicity of Aß(1-42). Aß oligomers bind to calmodulin (CaM) and calbindin-D28k with dissociation constants in the nanomolar Aß(1-42) concentration range. Aß and histidine-rich proteins have a high affinity for transition metal ions Cu2+, Fe3+ and Zn2+. In this work, we show that the fluorescence of Aß(1-42) HiLyteTM-Fluor555 can be used to monitor hexa-histidine peptide (His6) interaction with Aß(1-42). The formation of His6/Aß(1-42) complexes is also supported by docking results yielded by the MDockPeP Server. Also, we found that micromolar concentrations of His6 block the increase in the fluorescence of Aß(1-42) HiLyteTM-Fluor555 produced by its interaction with the proteins CaM and calbindin-D28k. In addition, we found that the His6-tag provides a high-affinity site for the binding of Aß(1-42) and Aß(25-35) peptides to the human recombinant cytochrome b5 reductase, and sensitizes this enzyme to inhibition by these peptides. In conclusion, our results suggest that a His6-tag could provide a valuable new tool to experimentally direct the action of neurotoxic Aß peptides toward selected cellular targets.

Kaempferol prevents the activation of complement C3 protein and the generation of reactive A1 astrocytes that mediate rat brain degeneration induced by 3-nitropropionic acid.

Kaempferol is a natural antioxidant present in vegetables and fruits used in human nutrition. In previous work, we showed that intraperitoneal (i.p.) kaempferol administration strongly protects against striatum neurodegeneration induced by i.p. injections of 3-nitropropionic acid (NPA), an animal model of Huntington's disease. Recently, we have shown that reactive A1 astrocytes generation is an early event in the neurodegeneration induced by NPA i.p. injections. In the present work, we have experimentally evaluated the hypothesis that kaempferol protects both against the activation of complement C3 protein and the generation of reactive A1 astrocytes in rat brain striatum and hippocampus. To this end, we have administered NPA and kaempferol i.p. injections to adult Wistar rats following the protocol described in previous work. Kaempferol administration prevents proteolytic activation of complement C3 protein and generation of reactive A1 astrocytes NPA-induced in the striatum and hippocampus. Also, it blocked the NPA-induced increase of NF-κB expression and enhanced secretion of cytokines IL-1α, TNFα, and C1q, which have been linked to the generation of reactive A1 astrocytes. In addition, kaempferol administration prevented the enhanced production of amyloid ß peptides in the striatum and hippocampus, a novel finding in NPA-induced brain degeneration found in this work.

Design and Experimental Evaluation of a Peptide Antagonist against Amyloid ß(1-42) Interactions with Calmodulin and Calbindin-D28k.

Amyloid ß1-42 (Aß(1-42)) oligomers have been linked to the pathogenesis of Alzheimer's disease (AD). Intracellular calcium (Ca2+) homeostasis dysregulation with subsequent alterations of neuronal excitability has been proposed to mediate Aß neurotoxicity in AD. The Ca2+ binding proteins calmodulin (CaM) and calbindin-D28k, whose expression levels are lowered in human AD brains, have relevant roles in neuronal survival and activity. In previous works, we have shown that CaM has a high affinity for Aß(1-42) oligomers and extensively binds internalized Aß(1-42) in neurons. In this work, we have designed a hydrophobic peptide of 10 amino acid residues: VFAFAMAFML (amidated-C-terminus amino acid) mimicking the interacting domain of CaM with Aß (1-42), using a combined strategy based on the experimental results obtained for Aß(1-42) binding to CaM and in silico docking analysis. The increase in the fluorescence intensity of Aß(1-42) HiLyteTM-Fluor555 has been used to monitor the kinetics of complex formation with CaM and with calbindin-D28k. The complexation between nanomolar concentrations of Aß(1-42) and calbindin-D28k is also a novel finding reported in this work. We found that the synthetic peptide VFAFAMAFML (amidated-C-terminus amino acid) is a potent inhibitor of the formation of Aß(1-42):CaM and of Aß(1-42):calbindin-D28k complexes.

The Relevance of Amyloid ß-Calmodulin Complexation in Neurons and Brain Degeneration in Alzheimer's Disease.

Intraneuronal amyloid ß (Aß) oligomer accumulation precedes the appearance of amyloid plaques or neurofibrillary tangles and is neurotoxic. In Alzheimer's disease (AD)-affected brains, intraneuronal Aß oligomers can derive from Aß peptide production within the neuron and, also, from vicinal neurons or reactive glial cells. Calcium homeostasis dysregulation and neuronal excitability alterations are widely accepted to play a key role in Aß neurotoxicity in AD. However, the identification of primary Aß-target proteins, in which functional impairment initiating cytosolic calcium homeostasis dysregulation and the critical point of no return are still pending issues. The micromolar concentration of calmodulin (CaM) in neurons and its high affinity for neurotoxic Aß peptides (dissociation constant ≈ 1 nM) highlight a novel function of CaM, i.e., the buffering of free Aß concentrations in the low nanomolar range. In turn, the concentration of Aß-CaM complexes within neurons will increase as a function of time after the induction of Aß production, and free Aß will rise sharply when accumulated Aß exceeds all available CaM. Thus, Aß-CaM complexation could also play a major role in neuronal calcium signaling mediated by calmodulin-binding proteins by Aß; a point that has been overlooked until now. In this review, we address the implications of Aß-CaM complexation in the formation of neurotoxic Aß oligomers, in the alteration of intracellular calcium homeostasis induced by Aß, and of dysregulation of the calcium-dependent neuronal activity and excitability induced by Aß.

Binding of Amyloid ß(1-42)-Calmodulin Complexes to Plasma Membrane Lipid Rafts in Cerebellar Granule Neurons Alters Resting Cytosolic Calcium Homeostasis.

Lipid rafts are a primary target in studies of amyloid ß (Aß) cytotoxicity in neurons. Exogenous Aß peptides bind to lipid rafts, which in turn play a key role in Aß uptake, leading to the formation of neurotoxic intracellular Aß aggregates. On the other hand, dysregulation of intracellular calcium homeostasis in neurons has been observed in Alzheimer's disease (AD). In a previous work, we showed that Aß(1-42), the prevalent Aß peptide found in the amyloid plaques of AD patients, binds with high affinity to purified calmodulin (CaM), with a dissociation constant ≈1 nM. In this work, to experimentally assess the Aß(1-42) binding capacity to intracellular CaM, we used primary cultures of mature cerebellar granule neurons (CGN) as a neuronal model. Our results showed a large complexation of submicromolar concentrations of Aß(1-42) dimers by CaM in CGN, up to 120 ± 13 picomoles of Aß(1-42) /2.5 × 106 cells. Using fluorescence microscopy imaging, we showed an extensive co-localization of CaM and Aß(1-42) in lipid rafts in CGN stained with up to 100 picomoles of Aß(1-42)-HiLyteTM-Fluor555 monomers. Intracellular Aß(1-42) concentration in this range was achieved by 2 h incubation of CGN with 2 µM Aß(1-42), and this treatment lowered the resting cytosolic calcium of mature CGN in partially depolarizing 25 mM potassium medium. We conclude that the primary cause of the resting cytosolic calcium decrease is the inhibition of L-type calcium channels of CGN by Aß(1-42) dimers, whose activity is inhibited by CaM:Aß(1-42) complexes bound to lipid rafts.

Early Reactive A1 Astrocytes Induction by the Neurotoxin 3-Nitropropionic Acid in Rat Brain.

3-Nitropropionic acid (NPA) administration to rodents produces degeneration of the striatum, accompanied by neurological disturbances that mimic Huntington's disease (HD) motor neurological dysfunctions. It has been shown that inflammation mediates NPA-induced brain degeneration, and activated microglia secreting cytokines interleukin-1α (IL-1α) and tumor necrosis factor α (TNFα) can induce a specific type of reactive neurotoxic astrocytes, named A1, which have been detected in post-mortem brain samples of Huntington's, Alzheimer's, and Parkinson's diseases. In this work we used an experimental model based on the intraperitoneal (i.p.) administration of NPA to adult Wistar rats at doses that can elicit extensive brain degeneration, and brain samples were taken before and after extensive brain damage monitored using 2,3,5-triphenyltetrazolium chloride (TTC) staining. Western blots and immunohistochemistry of brain slices show that i.p. NPA injections elicit significant increase in the expression levels of C3α subunit, a marker of generation of neurotoxic A1 astrocytes, and of cytokines IL-1α, TNFα, and C1q within the striatum, hippocampus, and cerebellum before the appearance of the HD-related neurological dysfunctions and neuronal death induced by NPA. Noteworthy, NPA administration primarily induces the generation of A1 astrocytes in the more recent phylogenetic area of the rat cerebellum. We conclude that the activation of complement C3 protein in the brain from Wistar rats is an early event in NPA-induced brain neurodegeneration.

Nicotine promotes neuron survival and partially protects from Parkinson's disease by suppressing SIRT6.

Parkinson's disease is characterized by progressive death of dopaminergic neurons, leading to motor and cognitive dysfunction. Epidemiological studies consistently show that the use of tobacco reduces the risk of Parkinson's. We report that nicotine reduces the abundance of SIRT6 in neuronal culture and brain tissue. We find that reduction of SIRT6 is partly responsible for neuroprotection afforded by nicotine. Additionally, SIRT6 abundance is greater in Parkinson's patient brains, and decreased in the brains of tobacco users. We also identify SNPs that promote SIRT6 expression and simultaneously associate with an increased risk of Parkinson's. Furthermore, brain-specific SIRT6 knockout mice are protected from MPTP-induced Parkinson's, while SIRT6 overexpressing mice develop more severe pathology. Our data suggest that SIRT6 plays a pathogenic and pro-inflammatory role in Parkinson's and that nicotine can provide neuroprotection by accelerating its degradation. Inhibition of SIRT6 may be a promising strategy to ameliorate Parkinson's and neurodegeneration.