Marcelo A. Chacón

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.

β-sheet breaker peptide prevents Aβ-induced spatial memory impairments with partial reduction of amyloid deposits

Current evidence supports the notion that β-amyloid deposits or Aβ intermediates may be responsible for the pathogenesis in Alzheimers disease (AD) patients. In the present work, we have assessed the neuroprotective effect of the chronic intraperitoneal administration of a five-amino-acid β-sheet breaker peptide (iAβ5p) on the rat behavioral deficit induced by the intrahippocampal Aβ-fibrils injection. At 1 month after the injection, animals showed a partial reduction of the amyloid deposits formed and a decreased astrocytic response around the injection site. More importantly, we report that following the iAβ5p treatment, hippocampal-dependent spatial learning paradigms, including the standard Morris water maze and a working memory analysis, showed a significant prevention from impairments induced by Aβ deposits in the dorsal hippocampus. Thus, it is possible that a noninvasive treatment such as the one presented here with β-sheet breaker peptides may be used as a potential therapy for AD patients.

Acetylcholinesterase-Aβ Complexes Are More Toxic than Aβ Fibrils in Rat Hippocampus: Effect on Rat β-Amyloid Aggregation, Laminin Expression, Reactive Astrocytosis, and Neuronal Cell Loss

Neuropathological changes generated by human amyloid-β peptide (Aβ) fibrils and Aβ-acetylcholinesterase (Aβ-AChE) complexes were compared in rat hippocampus in vivo. Results showed that Aβ-AChE complexes trigger a more dramatic response in situ than Aβ fibrils alone as characterized by the following features observed 8 weeks after treatment: 1) amyloid deposits were larger than those produced in the absence of AChE. In fact, AChE strongly stimulates rat Aβ aggregation in vitro as shown by turbidity measurements, Congo Red binding, as well as electron microscopy, suggesting that Aβ-AChE deposits observed in vivo probably recruited endogenous Aβ peptide; 2) the appearance of laminin expressing neurons surrounding Aβ-AChE deposits (such deposits are resistant to disaggregation by laminin in vitro); 3) an extensive astrocytosis revealed by both glial fibrillary acidic protein immunoreactivity and number counting of reactive hypertrophic astrocytes; and 4) a stronger neuronal cell loss in comparison with Aβ-injected animals. We conclude that the hippocampal injection of Aβ-AChE complexes results in the appearance of some features reminiscent of Alzheimer-like lesions in rat brain. Our studies are consistent with the notion that Aβ-AChE complexes are more toxic than Aβ fibrils and that AChE triggered some of the neurodegenerative changes observed in Alzheimers disease brains.

Human-like rodent amyloid-β-peptide determines Alzheimer pathology in aged wild-type Octodon degu

It is generally accepted that human Alzheimers disease (AD) neuropathology markers are completely absent in rodent brains. We report here that an aged wild-type South American rodent, Octodon degu, expresses neuronal beta-amyloid precursor protein (beta-APP695) displaying both intracellular and extracellular deposits of amyloid-beta-peptide (Abeta), intracellular accumulations of tau-protein and ubiquitin, a strong astrocytic response and acetylcholinesterase (AChE)-rich pyramidal neurons. The high amino acid homology (97.5%) between deguAbeta and humanAbeta sequences is probably a major factor in the appearance of AD markers in this aged rodent. Our results indicate that aged O. degu constitutes the first wild-type rodent model for neurodegenerative processes associated to AD.

Wnt signaling involvement in β-amyloid-dependent neurodegeneration

Abstract Alzheimer’s disease (AD) is a progressive dementia paralleled by selective neuronal death, which is probably caused by the cytotoxic effects of the amyloid-β peptide (Aβ). We have observed that Aβ-dependent neurotoxicity induces a loss of function of Wnt signaling components and that activation of this signaling cascade prevent such cytotoxic effects. Therefore we propose that compounds which mimic this signaling cascade may be candidates for therapeutic intervention in Alzheimer’s patients.

Frizzled‐1 is involved in the neuroprotective effect of Wnt3a against Aβ oligomers

The activation of the canonical Wnt signaling pathway protects hippocampal neurons against the toxicity of Alzheimers amyloid‐β‐peptide (Aβ), however, the role played by the Wnt receptors Frizzleds, has not been studied. We report here that Frizzled‐1 mediates the activation of the canonical Wnt/β‐catenin pathway by Wnt3a in PC12 cells. In addition, the protective effect of Wnt3a against the toxicity of Aβ oligomers was modulated by Frizzled‐1 expression levels in both PC12 cells and hippocampal neurons. Over‐expression of Frizzled‐1 significantly increased cell survival induced by Wnt3a and diminished caspase‐3 activation, while knocking‐down Frizzled‐1 expression by antisense oligonucleotides decreased the Wnt3a protection. Over‐expression of wild‐type β‐catenin, but not a transcriptionally inactive mutated version, prevented the toxicity of Aβ suggesting that the transcription of Wnt target genes may be involved in these events. This was confirmed by co‐transfecting both Frizzled‐1 and the inactive form of β‐catenin, which does not elicited protection levels similar to those showed with endogenous β‐catenin. Our results indicate that Wnt3a protects from Aβ‐oligomers toxicity by activating the canonical Wnt signaling pathway through the Frizzled‐1 receptor, suggesting a therapeutic potential for this signaling pathway in the treatment of Alzheimers disease. J. Cell. Physiol. 217: 215–227, 2008.

Acetylcholinesterase induces neuronal cell loss, astrocyte hypertrophy and behavioral deficits in mammalian hippocampus.

Previous studies have demonstrated that acetylcholinesterase (AChE) promotes the assembly of amyloid‐β‐peptides into neurotoxic amyloid fibrils and is toxic for chick retina neuronal cultures and neuroblastoma cells. Moreover, AChE is present in senile plaques in Alzheimers disease (AD) brains. Here we have studied the effect of AChE on astrocytes and hippocampal neurons in vivo. Morphological as well as behavioral disturbances were analyzed after intrahippocampal injection of AChE. Rats were trained in the Morris water maze and assayed for behavioral parameters. Neuronal cell loss was found in the upper leaf of the dentate gyrus in rats injected with AChE in comparison with control animals. Glial fibrillary acidic protein immunoreactivity showed astrocytic hypertrophy and the magnitude of the response was associated with neuronal cell loss. Behavioral results show that injection of AChE produces cognitive impairment demonstrated by an altered water maze performance including (i) a higher escape latency score, (ii) a decreased spatial acuity and (iii) a shorter time of swimming in the platform quadrant. These findings indicate that a local increment in neuronal AChE concentration at the mammalian hippocampus, such as those present in amyloid deposits, may play a role in triggering neuropathological and behavioral changes such as those observed in AD brains.

The N-terminal copper-binding domain of the amyloid precursor protein protects against Cu2+ neurotoxicity in vivo

The amyloid precursor protein (APP) contains a Cu binding domain (CuBD) localized between amino acids 135 and 156 (APP135‐156), which can reduce Cu2+ to Cu1+ in vitro. The physiological function of this APP domain has not yet being established; nevertheless several studies support the notion that the CuBD of APP is involved in Cu homeostasis. We used APP synthetic peptides to evaluate their protective properties against Cu2+ neurotoxicity in a bilateral intra‐hippocampal injection model. We found that human APP135‐156 protects against Cu2+‐induced neurotoxic effects, such as, impairment of spatial memory, neuronal cell loss, and astrogliosis. APP135‐156 lacking two histidine residues showed protection against Cu2+; however, APP135‐156 mutated in cysteine 144, a key residue in the reduction of Cu2+ to Cu1+, did not protect against Cu2+ neurotoxicity. In accordance with recent reports, the CuBD of the Caenorhabditis elegans, APL‐1, protected against Cu2+ neurotoxicity in vivo. We also found that Cu2+ neurotoxicity is associated with an increase in nitrotyrosine immunofluorescence as well as with a decrease in Cu2+ uptake. The CuBD of APP therefore may play a role in the detoxification of brain Cu.

A human prion protein peptide (PrP59-91) protects against copper neurotoxicity

Human cellular prion protein (PrPC) is involved in several neurodegenerative disorders; however, its normal function is unknown. We report here that a synthetic peptide corresponding to the four-octarepeat sequence of the PrPC (PrP59–91) protects hippocampal neurons against copper neurotoxic effects in vivo. Using a rat bilateral intrahippocampal injection model, we found that PrP59–91 protects against copper-induced neurotoxicity, including a recovery in spatial learning performance and a reduced neuronal cell loss and astrogliosis. Previous studies from our laboratory indicated that a tryptophan (Trp) residue plays a key role in the reduction of copper(II) to copper(I); therefore several PrP59–91 fragments lacking histidine (His) and Trp residues were tested for their capacity to protect from copper toxicity. A PrP59–91 peptide lacking His residue shows as much neuroprotection as the native peptide; however, PrP59–91 without Trp residues only partially protected against copper toxicity. The neuroprotective effect not only occurs with PrP59–91, in fact a full neuroprotection was also observed using just one octamer of the N-terminal region of prion protein. We conclude that the N-terminal tandem octarepeat of the human PrPC protects neurons against copper toxicity by a differential contribution of the binding (His) and reducing (Trp) copper activities of PrP59–91. Our results are consistent with the idea that PrPC function is related to copper homeostasis.

The functional links between prion protein and copper

Prion diseases are fatal neurodegenerative disorders associated with the conversion of the cellular prion protein (PrPC) into a pathologic isoform. Although the physiological function of PrPC remains unknown, evidence relates PrPC to copper metabolism and oxidative stress as suggested by its copper-binding properties in the N-terminal octapeptide repeat region. This region also reduces copper ions in vitro, and this reduction ability is associated with the neuroprotection exerted by the octarepeat region against copper in vivo. In addition, the promoter region of the PrPC gene contains putative metal response elements suggesting it may be regulated by heavy metals. Here we address some of the evidence that support a physiological link between PrPC and copper. Also, in vivo experiments suggesting the physiological relevance of PrPC interaction with heparan sulfate proteoglycans are discussed.