Alejandra R. Alvarez

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.

Protein kinase C inhibits amyloid β peptide neurotoxicity by acting on members of the Wnt pathway

Current evidence supports the notion that the amyloid β‐peptide (Aβ) plays a major role in the neurotoxicity observed in the brain in Alzheimers disease. However, the signal transduction mechanisms involved still remain unknown. In the present work, we analyzed the effect of protein kinase C (PKC) on some members of the Wnt signaling pathway and its implications for Aβ neurotoxicity. Activation of PKC by phorbol 12‐myristate 13‐acetate protected rat hippocampal neurons from Aβ toxicity. This effect was accomplished by inhibition of glycogen synthase kinase‐3β (GSK‐3β) activity, which led to the accumulation of cytoplasmic β‐catenin and transcriptional activation via β‐catenin/T‐cell factor/lymphoid enhancer factor‐1 (TCF/LEF1) of Wnt target genes, which in the present study were engrailed‐1 (en‐1) and cyclin D1 (cycD1). In contrast, inhibition of Ca2+‐dependent PKC isoforms activated GSK‐3β and offered no protection from Aβ neurotoxicity. Wnt‐3a and lithium salts, classical activators of the Wnt pathway, mimicked PKC activation. Our results suggest that regulation of members of the Wnt signaling pathway by Ca2+‐dependent PKC isoforms may be important in controlling the neurotoxic process induced by Aβ.

Amyloid–cholinesterase interactions

Acetylcholinesterase is an enzyme associated with senile plaques. Biochemical studies have indicated that acetylcholinesterase induces amyloid fibril formation by interaction throughout the peripherical anionic site of the enzyme forming highly toxic acetylcholinesterase–amyloid‐β peptide (Aβ) complexes. The pro‐aggregating acetylcholinesterase effect is associated with the intrinsic amyloidogenic properties of the corresponding Aβ peptide. The neurotoxicity induced by acetylcholinesterase–Aβ complexes is higher than the that induced by the Aβ peptide alone, both in vitro and in vivo. The fact that acetylcholinesterase accelerates amyloid formation and the effect is sensitive to peripherical anionic site blockers of the enzyme, suggests that specific and new acetylcholinesterase inhibitors may well provide an attractive possibility for treating Alzheimer’s disease. Recent studies also indicate that acetylcholinesterase induces the aggregation of prion protein with a similar dependence on the peripherical anionic site.

STI571 prevents apoptosis, tau phosphorylation and behavioural impairments induced by Alzheimer's β-amyloid deposits

There is evidence that amyloid beta-protein (Abeta) deposits or Abeta intermediates trigger pathogenic factors in Alzheimers disease patients. We have previously reported that c-Abl kinase activation involved in cell signalling regulates the neuronal death response to Abeta fibrils (Abeta(f)). In the present study we investigated the therapeutic potential of the selective c-Abl inhibitor STI571 on both the intrahippocampal injection of Abeta(f) and APPsw/PSEN1DeltaE9 transgenic mice Alzheimers disease models. Injection of Abeta(f) induced an increase in the numbers of p73 and c-Abl immunoreactive cells in the hippocampal area near to the lesion. Chronic intraperitoneal administration of STI571 reduced the rat behavioural deficit induced by Abeta(f), as well as apoptosis and tau phosphorylation. Our in vitro studies suggest that inhibition of the c-Abl/p73 signalling pathway is the mechanism underlying of the effects of STI571 on Abeta-induced apoptosis for the following reasons: (i) Abeta(f) induces p73 phosphorylation, the TAp73 isoform levels increase so as to enhance its proapoptotic function, and all these effects where reduced by STI571; (ii) c-Abl kinase activity is required for neuronal apoptosis and (iii) STI571 prevents the Abeta-induced increase in the expression of apoptotic genes. Furthermore, in the Abeta-injected area there was a huge increase in phosphorylated p73 and a larger number of TAp73-positive cells, with these changes being prevented by STI571 coinjection. Moreover, the intraperitoneal administration of STI571 rescued the cognitive decline in APPsw/PSEN1DeltaE9 mice, p73 phosphorylation, tau phosphorylation and caspase-3 activation in neurons around Abeta deposits. Besides, we observed a decrease in the number and size of Abeta deposits in the APPsw/PSEN1DeltaE9-STI571-treated mice. These results are consistent with the role of the c-Abl/p73 signalling pathway in Abeta neurodegeneration, and suggest that STI571-like compounds would be effective in therapeutic treatments of Alzheimer disease.

Reactive oxygen species trigger motoneuron death in non-cell-autonomous models of ALS through activation of c-Abl signaling

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease in which pathogenesis and death of motor neurons are triggered by non-cell-autonomous mechanisms. We showed earlier that exposing primary rat spinal cord cultures to conditioned media derived from primary mouse astrocyte conditioned media (ACM) that express human SOD1G93A (ACM-hSOD1G93A) quickly enhances Nav channel-mediated excitability and calcium influx, generates intracellular reactive oxygen species (ROS), and leads to death of motoneurons within days. Here we examined the role of mitochondrial structure and physiology and of the activation of c-Abl, a tyrosine kinase that induces apoptosis. We show that ACM-hSOD1G93A, but not ACM-hSOD1WT, increases c-Abl activity in motoneurons, interneurons and glial cells, starting at 60 min; the c-Abl inhibitor STI571 (imatinib) prevents this ACM-hSOD1G93A-mediated motoneuron death. Interestingly, similar results were obtained with ACM derived from astrocytes expressing SOD1G86R or TDP43A315T. We further find that co-application of ACM-SOD1G93A with blockers of Nav channels (spermidine, mexiletine, or riluzole) or anti-oxidants (Trolox, esculetin, or tiron) effectively prevent c-Abl activation and motoneuron death. In addition, ACM-SOD1G93A induces alterations in the morphology of neuronal mitochondria that are related with their membrane depolarization. Finally, we find that blocking the opening of the mitochondrial permeability transition pore with cyclosporine A, or inhibiting mitochondrial calcium uptake with Ru360, reduces ROS production and c-Abl activation. Together, our data point to a sequence of events in which a toxic factor(s) released by ALS-expressing astrocytes rapidly induces hyper-excitability, which in turn increases calcium influx and affects mitochondrial structure and physiology. ROS production, mediated at least in part through mitochondrial alterations, trigger c-Abl signaling and lead to motoneuron death.

Role of the Wnt receptor Frizzled-1 in presynaptic differentiation and function

BackgroundThe Wnt signaling pathway regulates several fundamental developmental processes and recently has been shown to be involved in different aspects of synaptic differentiation and plasticity. Some Wnt signaling components are localized at central synapses, and it is thus possible that this pathway could be activated at the synapse.ResultsWe examined the distribution of the Wnt receptor Frizzled-1 in cultured hippocampal neurons and determined that this receptor is located at synaptic contacts co-localizing with presynaptic proteins. Frizzled-1 was found in functional synapses detected with FM1-43 staining and in synaptic terminals from adult rat brain. Interestingly, overexpression of Frizzled-1 increased the number of clusters of Bassoon, a component of the active zone, while treatment with the extracellular cysteine-rich domain (CRD) of Frizzled-1 decreased Bassoon clustering, suggesting a role for this receptor in presynaptic differentiation. Consistent with this, treatment with the Frizzled-1 ligand Wnt-3a induced presynaptic protein clustering and increased functional presynaptic recycling sites, and these effects were prevented by co-treatment with the CRD of Frizzled-1. Moreover, in synaptically mature neurons Wnt-3a was able to modulate the kinetics of neurotransmitter release.ConclusionOur results indicate that the activation of the Wnt pathway through Frizzled-1 occurs at the presynaptic level, and suggest that the synaptic effects of the Wnt signaling pathway could be modulated by local activation through synaptic Frizzled receptors.

Imatinib therapy blocks cerebellar apoptosis and improves neurological symptoms in a mouse model of Niemann-Pick type C disease

Niemann‐Pick type C (NPC) disease is a fatal autosomal recessive disorder characterized by the accumulation of free cholesterol and glycosphingolipids in the endosomal‐lysosomal system. Patients with NPC disease have markedly progressive neuronal loss, mainly of cerebellar Purkinje neurons. There is strong evidence indicating that cholesterol accumulation and trafficking defects activate apoptosis in NPC brains. The purpose of this study was to analyze the relevance of apoptosis and particularly the proapoptotic c‐Abl/ p73 system in cerebellar neuron degeneration in NPC disease. We used the NPC1 mouse model to evaluate c‐Abl/p73 expression and activation in the cerebellum and the effect of therapy with the c‐Abl‐specific inhibitor imatinib. The proapoptotic c‐Abl/p73 system and the p73 target genes are expressed in the cerebellums of NPC mice. Furthermore, inhibition of c‐Abl with imatinib preserved Purkinje neurons and reduced general cell apoptosis in the cerebellum, improved neurological symptoms, and increased the survival of NPC mice. Moreover, this prosurvival effect correlated with reduced mRNA levels of p73 proapoptotic target genes. Our results suggest that the c‐Abl/p73 pathway is involved in NPC neurodegeneration and show that treatment with c‐Abl inhibitors is useful in delaying progressive neurodegeneration, supporting the use of imatinib for clinical treatment of patients with NPC disease.—Alvarez, A. R., Klein, A., Castro, J., Cancino, G. I., Amigo, J., Mosqueira, M., Vargas, L. M., Yévenes, L. F., Bronfman, F. C., Zanlungo, S. Imatinib therapy blocks cerebellar apoptosis and improves neurological symptoms in a mouse model of Niemann Pick type C disease. FASEB J. 22, 3617–3627 (2008)

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.

Laminin inhibits amyloid-β-peptide fibrillation

Abstract Laminin, an important extracellular matrix component is induced by brain injury and colocalizes with amyloid-β-peptide (Aβ) deposits in Alzheimer brains. We report here that laminin inhibits amyloid fibril formation as determined by thioflavin T fluorescence spectroscopy and electron microscopic examination. The inhibition of amyloid formation by laminin was concentration dependent and was observed at a laminin concentration of 300 nM, corresponding to a laminin/Aβ protein molar ratio of 1:800. The potential effect of laminin, may prove important to inhibit Aβ fibrillogenesis in vivo, specifically at the level of cerebral blood vessels.

c-Abl tyrosine kinase modulates tau pathology and Cdk5 phosphorylation in AD transgenic mice

The c-Abl tyrosine kinase is an important link in signal transduction pathways that promote cytoskeletal rearrangement and apoptotic signalling. We have previously shown that amyloid-β-peptide (Aβ) activates c-Abl. Herein we show that c-Abl participates in Aβ-induced tau phosphorylation through Cdk5 activation. We found that intraperitoneal administration of STI571, a specific inhibitor for c-Abl kinase, decreased tau phosphorylation in the APPswe/PSEN1ΔE9 transgenic mouse brain. In addition, when neurons were treated with Aβ we observed: (i) an increase in active c-Abl and tau phosphorylation, (ii) the prevention of tau phosphorylation by STI571 and (iii) the inhibition of c-Abl expression by shRNA, as well as the expression of a c-Abl kinase death mutant, decreased AT8 and PHF1 signals. Furthermore, the increase of c-Abl was associated with Tyr15 phosphorylation of Cdk5 and its association with c-Abl. Brains from APPswe/PSEN1ΔE9 mice showed higher levels of c-Abl and phospho-Cdk5 than wild-type mice. Moreover, STI571 treatment decreased the phospho-Cdk5 levels. Together, the evidence suggests that activation of c-Abl by Aβ promotes tau phosphorylation through Tyr15 phosphorylation-mediated Cdk5 activation.