Jordano Brito-Moreira

An anti-diabetes agent protects the mouse brain from defective insulin signaling caused by Alzheimer’s disease–associated Aβ oligomers

Defective brain insulin signaling has been suggested to contribute to the cognitive deficits in patients with Alzheimers disease (AD). Although a connection between AD and diabetes has been suggested, a major unknown is the mechanism(s) by which insulin resistance in the brain arises in individuals with AD. Here, we show that serine phosphorylation of IRS-1 (IRS-1pSer) is common to both diseases. Brain tissue from humans with AD had elevated levels of IRS-1pSer and activated JNK, analogous to what occurs in peripheral tissue in patients with diabetes. We found that amyloid-β peptide (Aβ) oligomers, synaptotoxins that accumulate in the brains of AD patients, activated the JNK/TNF-α pathway, induced IRS-1 phosphorylation at multiple serine residues, and inhibited physiological IRS-1pTyr in mature cultured hippocampal neurons. Impaired IRS-1 signaling was also present in the hippocampi of Tg mice with a brain condition that models AD. Importantly, intracerebroventricular injection of Aβ oligomers triggered hippocampal IRS-1pSer and JNK activation in cynomolgus monkeys. The oligomer-induced neuronal pathologies observed in vitro, including impaired axonal transport, were prevented by exposure to exendin-4 (exenatide), an anti-diabetes agent. In Tg mice, exendin-4 decreased levels of hippocampal IRS-1pSer and activated JNK and improved behavioral measures of cognition. By establishing molecular links between the dysregulated insulin signaling in AD and diabetes, our results open avenues for the investigation of new therapeutics in AD.

TNF-α mediates PKR-dependent memory impairment and brain IRS-1 inhibition induced by Alzheimer's β-amyloid oligomers in mice and monkeys

Alzheimers disease (AD) and type 2 diabetes appear to share similar pathogenic mechanisms. dsRNA-dependent protein kinase (PKR) underlies peripheral insulin resistance in metabolic disorders. PKR phosphorylates eukaryotic translation initiation factor 2α (eIF2α-P), and AD brains exhibit elevated phospho-PKR and eIF2α-P levels. Whether and how PKR and eIF2α-P participate in defective brain insulin signaling and cognitive impairment in AD are unknown. We report that β-amyloid oligomers, AD-associated toxins, activate PKR in a tumor necrosis factor α (TNF-α)-dependent manner, resulting in eIF2α-P, neuronal insulin receptor substrate (IRS-1) inhibition, synapse loss, and memory impairment. Brain phospho-PKR and eIF2α-P were elevated in AD animal models, including monkeys given intracerebroventricular oligomer infusions. Oligomers failed to trigger eIF2α-P and cognitive impairment in PKR(-/-) and TNFR1(-/-) mice. Bolstering insulin signaling rescued phospho-PKR and eIF2α-P. Results reveal pathogenic mechanisms shared by AD and diabetes and establish that proinflammatory signaling mediates oligomer-induced IRS-1 inhibition and PKR-dependent synapse and memory loss.

N‐Methyl‐d‐aspartate receptors are required for synaptic targeting of Alzheimer’s toxic amyloid‐β peptide oligomers

J. Neurochem. (2010) 115, 1520–1529.

Deregulation of excitatory neurotransmission underlying synapse failure in Alzheimer's disease

Alzheimer′s disease (AD) is the most common form of dementia in the elderly. Memory loss in AD is increasingly attributed to soluble oligomers of the amyloid‐β peptide (AβOs), toxins that accumulate in AD brains and target particular synapses. Glutamate receptors appear to be centrally involved in synaptic targeting by AβOs. Once bound to neurons, AβOs dysregulate the activity and reduce the surface expression of both N‐methyl‐d‐aspartate (NMDA) and 2‐amino‐3‐(3‐hydroxy‐5‐methyl‐isoxazol‐4‐yl)propanoic acid (AMPA) types of glutamate receptors, impairing signaling pathways involved in synaptic plasticity. In the extracellular milieu, AβOs promote accumulation of the excitatory amino acids, glutamate and d‐serine. This leads to overactivation of glutamate receptors, triggering abnormal calcium signals with noxious impacts on neurons. Here, we review key findings linking AβOs to deregulated glutamate neurotransmission and implicating this as a primary mechanism of synapse failure in AD. We also discuss strategies to counteract the impact of AβOs on excitatory neurotransmission. In particular, we review evidence showing that inducing neuronal hyperpolarization via activation of inhibitory GABAA receptors prevents AβO‐induced excitotoxicity, suggesting that this could comprise a possible therapeutic approach in AD.

Activation of GABAA receptors by taurine and muscimol blocks the neurotoxicity of β-amyloid in rat hippocampal and cortical neurons

The beta-amyloid peptide (Abeta) is centrally related to the pathogenesis of Alzheimers disease (AD) and is potently neurotoxic to central nervous system neurons. The neurotoxicity of Abeta has been partially related to the over activation of glutamatergic transmission and excitotoxicity. Taurine is a naturally occurring beta-amino acid present in the mammalian brain. Due to its safety and tolerability, taurine has been clinically used in humans in the treatment of a number of non-neurological disorders. Here, we show that micromolar doses of taurine block the neurotoxicity of Abeta to rat hippocampal and cortical neurons in culture. Moreover, taurine also rescues central neurons from the excitotoxicity induced by high concentrations of extracellular glutamate. Neuroprotection by taurine is abrogated by picrotoxin, a GABA(A) receptor antagonist. GABA and muscimol, an agonist of the GABA(A) receptor, also block neuronal death induced by Abeta in rat hippocampal and cortical neurons. These results suggest that activation of GABA(A) receptors protects neurons against Abeta toxicity in AD-affected regions of the mammalian brain and that taurine should be investigated as a novel therapeutic tool in the treatment of AD and of other neurological disorders in which excitotoxicity plays a relevant role.

Alzheimer's Disease-Like Pathology Induced by Amyloid-β Oligomers in Nonhuman Primates

Alzheimers disease (AD) is a devastating neurodegenerative disorder and a major medical problem. Here, we have investigated the impact of amyloid-β (Aβ) oligomers, AD-related neurotoxins, in the brains of rats and adult nonhuman primates (cynomolgus macaques). Soluble Aβ oligomers are known to accumulate in the brains of AD patients and correlate with disease-associated cognitive dysfunction. When injected into the lateral ventricle of rats and macaques, Aβ oligomers diffused into the brain and accumulated in several regions associated with memory and cognitive functions. Cardinal features of AD pathology, including synapse loss, tau hyperphosphorylation, astrocyte and microglial activation, were observed in regions of the macaque brain where Aβ oligomers were abundantly detected. Most importantly, oligomer injections induced AD-type neurofibrillary tangle formation in the macaque brain. These outcomes were specifically associated with Aβ oligomers, as fibrillar amyloid deposits were not detected in oligomer-injected brains. Human and macaque brains share significant similarities in terms of overall architecture and functional networks. Thus, generation of a macaque model of AD that links Aβ oligomers to tau and synaptic pathology has the potential to greatly advance our understanding of mechanisms centrally implicated in AD pathogenesis. Furthermore, development of disease-modifying therapeutics for AD has been hampered by the difficulty in translating therapies that work in rodents to humans. This new approach may be a highly relevant nonhuman primate model for testing therapeutic interventions for AD.

Activation of D1/D5 dopamine receptors protects neurons from synapse dysfunction induced by amyloid-beta oligomers.

Soluble oligomers of the amyloid-β peptide (AβOs) accumulate in the brains of Alzheimer disease (AD) patients and are implicated in synapse failure and early memory loss in AD. AβOs have been shown to impact synapse function by inhibiting long term potentiation, facilitating the induction of long term depression and inducing internalization of both AMPA and NMDA glutamate receptors, critical players in plasticity mechanisms. Because activation of dopamine D1/D5 receptors plays important roles in memory circuits by increasing the insertion of AMPA and NMDA receptors at synapses, we hypothesized that selective activation of D1/D5 receptors could protect synapses from the deleterious action of AβOs. We show that SKF81297, a selective D1/D5 receptor agonist, prevented the reduction in surface levels of AMPA and NMDA receptors induced by AβOs in hippocampal neurons in culture. Protection by SKF81297 was abrogated by the specific D1/D5 antagonist, SCH23390. Levels of AMPA receptor subunit GluR1 phosphorylated at Ser845, which regulates AMPA receptor association with the plasma membrane, were reduced in a calcineurin-dependent manner in the presence of AβOs, and treatment with SKF81297 prevented this reduction. Establishing the functional relevance of these findings, SKF81297 blocked the impairment of long term potentiation induced by AβOs in hippocampal slices. Results suggest that D1/D5 receptors may be relevant targets for development of novel pharmacological approaches to prevent synapse failure in AD.

Human apolipoprotein A–I binds amyloid-β and prevents Aβ-induced neurotoxicity

Aggregates of the amyloid-beta peptide (Abeta) play a central role in the pathogenesis of Alzheimers disease (AD). Identification of proteins that physiologically bind Abeta and modulate its aggregation and neurotoxicity could lead to the development of novel disease-modifying approaches in AD. By screening a phage display peptide library for high affinity ligands of aggregated Abeta(1-42), we isolated a peptide homologous to a highly conserved amino acid sequence present in the N-terminus of apolipoprotein A-I (apoA-I). We show that purified human apoA-I and Abeta form non-covalent complexes and that interaction with apoA-I affects the morphology of amyloid aggregates formed by Abeta. Significantly, Abeta/apoA-I complexes were also detected in cerebrospinal fluid from AD patients. Interestingly, apoA-I and apoA-I-containing reconstituted high density lipoprotein particles protect hippocampal neuronal cultures from Abeta-induced oxidative stress and neurodegeneration. These results suggest that human apoA-I modulates Abeta aggregation and Abeta-induced neuronal damage and that the Abeta-binding domain in apoA-I may constitute a novel framework for the design of inhibitors of Abeta toxicity.

Inhibition of Choline Acetyltransferase as a Mechanism for Cholinergic Dysfunction Induced by Amyloid-β Peptide Oligomers

Background: Cholinergic dysfunction is an early feature of Alzheimer disease (AD). Results: Soluble oligomers of the amyloid-β peptide (Aβ) bind to cholinergic neurons and inhibit choline acetyltransferase (ChAT) activity before any cell death or lesion. Conclusion: ChAT inhibition might impair acetylcholine production and cholinergic function in AD brains. Significance: This novel effect of Aβ oligomers may be relevant in early stage AD pathology. Dysregulated cholinergic signaling is an early hallmark of Alzheimer disease (AD), usually ascribed to degeneration of cholinergic neurons induced by the amyloid-β peptide (Aβ). It is now generally accepted that neuronal dysfunction and memory deficits in the early stages of AD are caused by the neuronal impact of soluble Aβ oligomers (AβOs). AβOs build up in AD brain and specifically attach to excitatory synapses, leading to synapse dysfunction. Here, we have investigated the possibility that AβOs could impact cholinergic signaling. The activity of choline acetyltransferase (ChAT, the enzyme that carries out ACh production) was inhibited by ∼50% in cultured cholinergic neurons exposed to low nanomolar concentrations of AβOs. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction, lactate dehydrogenase release, and [3H]choline uptake assays showed no evidence of neuronal damage or loss of viability that could account for reduced ChAT activity under these conditions. Glutamate receptor antagonists fully blocked ChAT inhibition and oxidative stress induced by AβOs. Antioxidant polyunsaturated fatty acids had similar effects, indicating that oxidative damage may be involved in ChAT inhibition. Treatment with insulin, previously shown to down-regulate neuronal AβO binding sites, fully prevented AβO-induced inhibition of ChAT. Interestingly, we found that AβOs selectively bind to ∼50% of cultured cholinergic neurons, suggesting that ChAT is fully inhibited in AβO-targeted neurons. Reduction in ChAT activity instigated by AβOs may thus be a relevant event in early stage AD pathology, preceding the loss of cholinergic neurons commonly observed in AD brains.

Aβ Oligomers Induce Glutamate Release from Hippocampal Neurons

Soluble oligomers of the amyloid-β peptide (AβOs) accumulate in Alzheimers disease (AD) brain and have been implicated in mechanisms of pathogenesis. The neurotoxicity of AβOs appears to be, at least in part, due to dysregulation of glutamate signaling. Here, we show that AβOs promote extracellular accumulation of glutamate and d-serine, a co-agonist at glutamate receptors of the N-methyl-d-aspartate subtype (NMDARs), in hippocampal neuronal cultures. The increase in extracellular glutamate levels induced by AβOs was blocked by the sodium channel blocker tetrodotoxin (TTX), by the NMDAR blocker (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) and by removal of Ca(2+) from the extracellular medium, indicating dependence on excitatory neuronal activity. AβOs enhanced both the release of pre-synaptic vesicles labeled by FM1-43 and spontaneous post-synaptic activity measured by whole-cell patch-clamp. Activation of inhibitory GABA(A) receptors by taurine blocked the increase in extracellular glutamate levels, suggesting that selective pharmacological inhibition of neuronal activity can counteract the impact of AbOs on glutamate dyshomeostasis. Results reveal a novel mechanism by which Ab oligomers promote abnormal release of glutamate in hippocampal neurons, which may contribute to dysregulation of excitatory signaling in the brain.