Nibaldo C. Inestrosa

Acetylcholinesterase accelerates assembly of amyloid-β-peptides into Alzheimer's fibrils: Possible role of the peripheral site of the enzyme

Acetylcholinesterase (AChE), an important component of cholinergic synapses, colocalizes with amyloid-beta peptide (A beta) deposits of Alzheimers brain. We report here that bovine brain AChE, as well as the human and mouse recombinant enzyme, accelerates amyloid formation from wild-type A beta and a mutant A beta peptide, which alone produces few amyloid-like fibrils. The action of AChE was independent of the subunit array of the enzyme, was not affected by edrophonium, an active site inhibitor, but it was affected by propidium, a peripheral anionic binding site ligand. Butyrylcholinesterase, an enzyme that lacks the peripheral site, did not affect amyloid formation. Furthermore, AChE is a potent amyloid-promoting factor when compared with other A beta-associated proteins. Thus, in addition to its role in cholinergic synapses, AChE may function by accelerating A beta formation and could play a role during amyloid deposition in Alzheimers brain.

Metalloenzyme-like Activity of Alzheimer's Disease β-Amyloid Cu-DEPENDENT CATALYTIC CONVERSION OF DOPAMINE, CHOLESTEROL, AND BIOLOGICAL REDUCING AGENTS TO NEUROTOXIC H2O2

β-Amyloid (Aβ) 1–42, implicated in the pathogenesis of Alzheimers disease, forms an oligomeric complex that binds copper at a CuZn superoxide dismutase-like binding site. Aβ·Cu complexes generate neurotoxic H2O2 from O2 through Cu2+ reduction, but the reaction mechanism has been unclear. We now report that Aβ1–42, when binding up to 2 eq of Cu2+, generates the H2O2catalytically by recruiting biological reducing agents as substrates under conditions where the Cu2+ or reducing agents will not form H2O2 themselves. Cholesterol is an important substrate for this activity, as are vitamin C,l-DOPA, and dopamine (V maxfor dopamine = 34.5 nm/min, K m = 8.9 μm). The activity was inhibited by anti-Aβ antibodies, Cu2+ chelators, and Zn2+. Toxicity of Aβ in neuronal culture was consistent with catalytic H2O2 production. Aβ was not toxic in cell cultures in the absence of Cu2+, and dopamine (5 μm) markedly exaggerated the neurotoxicity of 200 nm Aβ1–42·Cu. Therefore, microregional catalytic H2O2 production, combined with the exhaustion of reducing agents, may mediate the neurotoxicity of Aβ in Alzheimers disease, and inhibitors of this novel activity may be of therapeutic value.

Emerging roles of Wnts in the adult nervous system

The roles of the Wnt signalling pathway in several developmental processes, including synaptic differentiation, are well characterized. The expression of Wnt ligands and Wnt signalling components in the mature mammalian CNS suggests that this pathway might also play a part in synaptic maintenance and function. In fact, Wnts have a crucial role in synaptic physiology, as they modulate the synaptic vesicle cycle, the trafficking of neurotransmitter receptors and the interaction of these receptors with scaffold proteins in postsynaptic regions. In addition, Wnts participate in adult neurogenesis and protect excitatory synaptic terminals from amyloid-β oligomer toxicity. Here, the latest insights into the function of Wnt signalling in the adult nervous system and therapeutic opportunities for neurodegenerative diseases such as Alzheimers and Parkinsons disease are discussed.

The role of oxidative stress in the toxicity induced by amyloid β-peptide in Alzheimer’s disease

One of the theories involved in the etiology of Alzheimers disease (AD) is the oxidative stress hypothesis. The amyloid beta-peptide (A beta), a hallmark in the pathogenesis of AD and the main component of senile plaques, generates free radicals in a metal-catalyzed reaction inducing neuronal cell death by a reactive oxygen species mediated process which damage neuronal membrane lipids, proteins and nucleic acids. Therefore, the interest in the protective role of different antioxidants in AD such as vitamin E, melatonin and estrogens is growing up. In this review we summarize data that support the involvement of oxidative stress as an active factor in A beta-mediated neuropathology, by triggering or facilitating neurodegeneration, through a wide range of molecular events that disturb neuronal cell homeostasis.

Activation of Wnt signaling rescues neurodegeneration and behavioral impairments induced by β -amyloid fibrils

Alzheimers disease (AD) is a progressive neurodegenerative disorder, which is probably caused by the cytotoxic effect of the amyloid β-peptide (Aβ). We report here molecular changes induced by Aβ, both in neuronal cells in culture and in rats injected in the dorsal hippocampus with preformed Aβ fibrils, as an in vivo model of the disease. Results indicate that in both systems, Aβ neurotoxicity resulted in the destabilization of endogenous levels of β-catenin, a key transducer of the Wnt signaling pathway. Lithium chloride, which mimics Wnt signaling by inhibiting glycogen synthase kinase-3β promoted the survival of post-mitotic neurons against Aβ neurotoxicity and recovered cytosolic β-catenin to control levels. Moreover, the neurotoxic effect of Aβ fibrils was also modulated with protein kinase C agonists/inhibitors and reversed with conditioned medium containing the Wnt-3a ligand. We also examined the spatial memory performance of rats injected with preformed Aβ fibrils in the Morris water maze paradigm, and found that chronic lithium treatment protected neurodegeneration by rescuing β-catenin levels and improved the deficit in spatial learning induced by Aβ. Our results are consistent with the idea that Aβ-dependent neurotoxicity induces a loss of function of Wnt signaling components and indicate that lithium or compounds that mimic this signaling cascade may be putative candidates for therapeutic intervention in Alzheimers patients.

Peroxisome Proliferator-activated Receptor γ Up-regulates the Bcl-2 Anti-apoptotic Protein in Neurons and Induces Mitochondrial Stabilization and Protection against Oxidative Stress and Apoptosis

Peroxisome proliferator-activated receptor γ (PPARγ) has been proposed as a therapeutic target for neurodegenerative diseases because of its anti-inflammatory action in glial cells. However, PPARγ agonists preventβ-amyloid (Aβ)-induced neurodegeneration in hippocampal neurons, and PPARγ is activated by the nerve growth factor (NGF) survival pathway, suggesting a neuroprotective anti-inflammatory independent action. Here we show that the PPARγ agonist rosiglitazone (RGZ) protects hippocampal and dorsal root ganglion neurons against Aβ-induced mitochondrial damage and NGF deprivation-induced apoptosis, respectively, and promotes PC12 cell survival. In neurons and in PC12 cells RGZ protective effects are associated with increased expression of the Bcl-2 anti-apoptotic protein. NGF-differentiated PC12 neuronal cells constitutively overexpressing PPARγ are resistant to Aβ-induced apoptosis and morphological changes and show functionally intact mitochondria and no increase in reactive oxygen species when challenged with up to 50 μm H2O2. Conversely, cells expressing a dominant negative mutant of PPARγ show increased Aβ-induced apoptosis and disruption of neuronal-like morphology and are highly sensitive to oxidative stress-induced impairment of mitochondrial function. Cells overexpressing PPARγ present a 4- to 5-fold increase in Bcl-2 protein content, whereas in dominant negative PPARγ-expressing cells, Bcl-2 is barely detected. Bcl-2 knockdown by small interfering RNA in cells overexpressing PPARγ results in increased sensitivity to Aβ and oxidative stress, further suggesting that Bcl-2 up-regulation mediates PPARγ protective effects. PPARγ prosurvival action is independent of the signal-regulated MAPK or the Akt prosurvival pathways. Altogether, these data suggest that PPARγ supports survival in neurons in part through a mechanism involving increased expression of Bcl-2.

Fatty acid oxidation by human liver peroxisomes

Abstract A cyanide insensitive fatty acid oxidation system is detected in human liver and is shown to be localized in peroxisomes by subcellular fractionation in Metrizamide continuous density gradients. Fatty acyl-CoA oxidase, its characteristic enzyme, acts maximally on C 12 –C 18 saturated fatty acids and on oleoyl-CoA and requires FAD. These results, together with the already established properties of the system in rat liver, support its potential contribution to lipid metabolism and to the hypolipidemic effect of Clofibrate and related drugs in humans.

Activation of Wnt signaling by lithium and rosiglitazone reduced spatial memory impairment and neurodegeneration in brains of an APPswe/PSEN1ΔE9 mouse model of Alzheimer's disease

Alzheimers disease (AD) is a neurodegenerative disorder characterized by a progressive deterioration of cognitive abilities, accumulation of the amyloid-β-peptide (Aβ) and synaptic alterations. Treatment with lithium has been shown to provide neuroprotection against several insults, including protection against Aβ neurotoxicity in vitro. Rosiglitazone, a peroxisome proliferator activated receptor-γ agonist, has been shown to attenuate Aβ-peptide neurotoxic effects, including the inflammatory response of microglia and astrocytes. Both types of drugs activate Wnt signaling, a pathway that has been shown to be related to AD. In this study, a double transgenic mouse model, which coexpresses APPswe and the exon 9 deletion of the presenilin 1 (PSEN1) gene, was used to examine, in vivo, the effect of lithium and rosiglitazone on Aβ neurotoxicity. Mice were tested for spatial memory, and their brain samples were used for histochemical and biochemical analysis. In this study, we report that both drugs significantly reduced (1) spatial memory impairment induced by amyloid burden; (2) Aβ aggregates and Aβ oligomers; and (3) astrocytic and microglia activation. They also prevented changes in presynaptic and postsynaptic marker proteins. Finally, both drugs activate Wnt signaling shown by the increase in β-catenin and by the inhibition of the glycogen synthase kinase-3β. We conclude that lithium and rosiglitazone, possibly by the activation of the Wnt signaling pathway, reduce various AD neuropathological markers and may be considered as potential therapeutic agents against the disease.

Wnt signaling in neuroprotection and stem cell differentiation.

In the past several years, we postulated that the loss of Wnt signaling was implicated in the pathology of Alzheimers disease (AD). Since then, our lab and other groups have confirmed the involvement of the Wnt signaling in some aspects of AD. So far, we have demonstrated that activation of Wnt signaling protects neurons against neurotoxic injuries, including both amyloid-beta (Abeta) fibrils and Abeta oligomers by using either lithium, an inhibitor of the glycogen-synthase-kinase-3beta (GSK-3beta), or different Wnt ligands. Also, we have found that several molecules which activate well known neurotransmitter systems and other signaling system, are able by crosstalk to activate Wnt/beta-catenin signaling in order to protect neurons against both Abeta fibrils or Abeta oligomers. In particular, the activation of non-canonical Wnt signaling was able to protect postsynaptic regions and dendritic spines against Abeta oligomers. Furthermore Wnt signaling ligands also affect stem cells, and they are also involved in cell fate decision during neurogenesis and embryonic development as well as in adult stem cells differentiation in the nervous system. The Wnt signaling plays a key role modulating their cell differentiation or proliferation states. Altogether, these findings in both stem cell biology and neuroprotection, may introduce new approaches in the treatment of neurodegenerative diseases, including drug screening and therapies against neurodegenerative diseases which activates the Wnt signaling pathway.

Wnt-7a Modulates the Synaptic Vesicle Cycle and Synaptic Transmission in Hippocampal Neurons

Wnt signaling is essential for neuronal development and the maintenance of the developing nervous system. Recent studies indicated that Wnt signaling modulates long term potentiation in adult hippocampal slices. We report here that different Wnt ligands are present in hippocampal neurons of rat embryo and adult rat, including Wnt-4, -5a, -7a, and -11. Wnt-7a acts as a canonical Wnt ligand in rat hippocampal neurons, stimulates clustering of presynaptic proteins, and induces recycling and exocytosis of synaptic vesicles as studied by FM dyes. Wnt-3a has a moderate effect on recycling of synaptic vesicles, and no effect of Wnt-1 and Wnt-5a was detected. Electrophysiological analysis on adult rat hippocampal slices indicates that Wnt-7a, but not Wnt-5a, increases neurotransmitter release in CA3-CA1 synapses by decreasing paired pulse facilitation and increasing the miniature excitatory post-synaptic currents frequency. These results indicate that the presynaptic function of rat hippocampal neurons is modulated by the canonical Wnt signaling.