Luis E. Santos

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.

Cross Talk Between Brain Innate Immunity and Serotonin Signaling Underlies Depressive-Like Behavior Induced by Alzheimer's Amyloid-β Oligomers in Mice

Considerable clinical and epidemiological evidence links Alzheimers disease (AD) and depression. However, the molecular mechanisms underlying this connection are largely unknown. We reported recently that soluble Aβ oligomers (AβOs), toxins that accumulate in AD brains and are thought to instigate synapse damage and memory loss, induce depressive-like behavior in mice. Here, we report that the mechanism underlying this action involves AβO-induced microglial activation, aberrant TNF-α signaling, and decreased brain serotonin levels. Inactivation or ablation of microglia blocked the increase in brain TNF-α and abolished depressive-like behavior induced by AβOs. Significantly, we identified serotonin as a negative regulator of microglial activation. Finally, AβOs failed to induce depressive-like behavior in Toll-like receptor 4-deficient mice and in mice harboring a nonfunctional TLR4 variant in myeloid cells. Results establish that AβOs trigger depressive-like behavior via a double impact on brain serotonin levels and microglial activation, unveiling a cross talk between brain innate immunity and serotonergic signaling as a key player in mood alterations in AD. SIGNIFICANCE STATEMENT Alzheimers disease (AD) is a progressive neurodegenerative disorder and the main cause of dementia in the world. Brain accumulation of amyloid-β oligomers (AβOs) is a major feature in the pathogenesis of AD. Although clinical and epidemiological data suggest a strong connection between AD and depression, the underlying mechanisms linking these two disorders remain largely unknown. Here, we report that aberrant activation of the brain innate immunity and decreased serotonergic tonus in the brain are key players in AβO-induced depressive-like behavior in mice. Our findings may open up new possibilities for the development of effective therapeutics for AD and depression aimed at modulating microglial function.

Prion protein modulates monoaminergic systems and depressive-like behavior in mice

Background: The prion protein (PrPC) functions as a scaffold for cell surface signaling systems, and plays a role in neurodegenerative diseases that include clinical depression among their symptoms. Results: PrPC-null mice showed depressive-like behavior concomitant with functional changes in monoaminergic systems. Conclusion: PrPC regulates functions of monoaminergic synapses. Significance: PrPC may be involved in major depression and related neuropsychiatric disorders. We sought to examine interactions of the prion protein (PrPC) with monoaminergic systems due to: the role of PrPC in both Prion and Alzheimer diseases, which include clinical depression among their symptoms, the implication of monoamines in depression, and the hypothesis that PrPC serves as a scaffold for signaling systems. To that effect we compared both behavior and monoaminergic markers in wild type (WT) and PrPC-null (PrP−/−) mice. PrP−/− mice performed poorly when compared with WT in forced swimming, tail suspension, and novelty suppressed feeding tests, typical of depressive-like behavior, but not in the control open field nor rotarod motor tests; cyclic AMP responses to stimulation of D1 receptors by dopamine was selectively impaired in PrP−/− mice, and responses to serotonin, but not to norepinephrine, also differed between genotypes. Contents of dopamine, tyrosine hydroxylase, and the 5-HT5A serotonin receptor were increased in the cerebral cortex of PrP−/−, as compared with WT mice. Microscopic colocalization, as well as binding in overlay assays were found of PrPC with both the 5HT5A and D1, but not D4 receptors. The data are consistent with the scaffolding of monoaminergic signaling modules by PrPC, and may help understand the pathogenesis of clinical depression and neurodegenerative disorders.

Secreted Human Amyloid Precursor Protein Binds Semaphorin 3a and Prevents Semaphorin-Induced Growth Cone Collapse

The amyloid precursor protein (APP) is well known for giving rise to the amyloid-β peptide and for its role in Alzheimers disease. Much less is known, however, on the physiological roles of APP in the development and plasticity of the central nervous system. We have used phage display of a peptide library to identify high-affinity ligands of purified recombinant human sAPPα695 (the soluble, secreted ectodomain from the main neuronal APP isoform). Two peptides thus selected exhibited significant homologies with the conserved extracellular domain of several members of the semaphorin (Sema) family of axon guidance proteins. We show that sAPPα695 binds both purified recombinant Sema3A and Sema3A secreted by transfected HEK293 cells. Interestingly, sAPPα695 inhibited the collapse of embryonic chicken (Gallus gallus domesticus) dorsal root ganglia growth cones promoted by Sema3A (Kd≤8·10−9 M). Two Sema3A-derived peptides homologous to the peptides isolated by phage display blocked sAPPα binding and its inhibitory action on Sema3A function. These two peptides are comprised within a domain previously shown to be involved in binding of Sema3A to its cellular receptor, suggesting a competitive mechanism by which sAPPα modulates the biological action of semaphorins.

Murine dopaminergic Müller cells restore motor function in a model of Parkinson's disease

Müller cells constitute the main glial cell type in the retina where it interacts with virtually all cells displaying relevant functions to retinal physiology. Under appropriate stimuli, Müller cells may undergo dedifferentiation, being able to generate other neural cell types. Here, we show that purified mouse Müller cells in culture express a group of proteins related to the dopaminergic phenotype, including the nuclear receptor‐related 1 protein, required for dopaminergic differentiation, as well the enzyme tyrosine hydroxylase. These dopaminergic components are active, since Müller cells are able to synthesize and release dopamine to the extracellular medium. Moreover, Müller‐derived tyrosine hydroxylase can be regulated, increasing its activity because of phosphorylation of serine residues in response to agents that increase intracellular cAMP levels. These observations were extended to glial cells obtained from adult monkey retinas with essentially the same results. To address the potential use of dopaminergic Müller cells as a source of dopamine in cell therapy procedures, we used a mouse model of Parkinsons disease, in which mouse Müller cells with the dopaminergic phenotype were transplanted into the striatum of hemi‐parkinsonian mice generated by unilateral injection of 6‐hydroxydopamine. These cells fully decreased the apomorphine‐induced rotational behavior and restored motor functions in these animals, as measured by the rotarod and the forelimb‐use asymmetry (cylinder) tests. The data indicate local restoration of dopaminergic signaling in hemi‐parkinsonian mice confirmed by measurement of striatal dopamine after Müller cell grafting.

Functional plasticity of GAT-3 in avian Müller cells is regulated by neurons via a glutamatergic input

GABA (γ-amino butyric acid) is the major inhibitory transmitter in the central nervous system and its action is terminated by specific transporters (GAT), found in neurons and glial cells. We have previously described that GAT-3 is responsible for GABA uptake activity in cultured avian Müller cells and that it operates in a Na(+) and Cl(-) dependent manner. Here we show that glutamate decreases [(3)H] GABA uptake in purified cultured glial cells up to 50%, without causing cell death. This effect is mediated by ionotropic glutamatergic receptors. Glutamate inhibition on GABA uptake is not reverted by inhibitors of protein kinase C or modified by agents that modulate cyclic AMP/PKA. Biotinylation experiments demonstrate that this reduction in GABA uptake correlates with a decrease in GAT-3 plasma membrane levels. Interestingly, both GAT-1 and GAT-3 mRNA levels are also decreased by glutamate. Conditioned media (CM) prepared from retinal neurons could also decrease GABA influx, and glutamate receptor antagonists (MK-801 + CNQX) were able to prevent this effect. However, glutamate levels in CM were not different from those found in fresh media, indicating that a glutamatergic co-agonist or modulator could be regulating GABA uptake by Müller cells in this scenario. In the whole avian retina, GAT-3 is present from embryonic day 5 (E5) increasing up to the end of embryonic development and post-hatch period exclusively in neuronal layers. However, this pattern may change in pathological conditions, which drive GAT-3 expression in Müller cells. Our data suggest that in purified cultures and upon extensive neuronal lesion in vivo, shown as a Brn3a reduced neuronal cells and an GFAP increased gliosis, Müller glia may change its capacity to take up GABA due to GAT-3 up regulation and suggests a regulatory interplay mediated by glutamate between neurons and glial cells in this process.

The Importance of Serotonin in Exercise-Induced Adult Neurogenesis: New Evidence from Tph2−/− Mice

The neurotransmitter serotonin (5-HT) is a well established modulator of adult neurogenesis. In the last decade, several lines of research involving pharmacological enhancement of 5-HT levels, depletion of serotonergic neurons and direct activation of 5-HT receptors have shown consistently that 5-HT

Pituitary adenylyl cyclase-activating polypeptide receptor re-sensitization induces plastic changes in the dopaminergic phenotype in the mature avian retina

Pituitary Adenylyl Cyclase‐Activating Polypeptide (PACAP) is a neuroactive peptide present in the avian retina where it activates adenylyl cyclase (AC) since early in development via PACAP receptors. The synthesis of cAMP in response to PACAP is observed since embryonic day 8/9 (E8/9). After E12, signaling via PACAP receptors desensitizes, reaching very low levels in the mature tissue. We show here that chronic administration of PACAP in vitro desensitizes PACAP‐induced cAMP accumulation, while the administration of the PACAP antagonist (PACAP 6‐38) re‐sensitizes PACAP receptor/cyclase system in vitro and in vivo. Moreover, a twofold increase in the number of tyrosine hydroxylase positive (TH+) cells is observed after in vivo injection of PACAP6‐38. NURR1, a transcription factor associated with the differentiation of dopaminergic cells in the CNS, is present in the chick retina in all developmental stages studied. The presence of NURR1 positive cells in the mature tissue far exceeds the number of TH+ cells, suggesting that these NURR1‐positive cells might have the potential to express the dopaminergic phenotype. Our data show that if PACAP signaling is increased in mature retinas, plastic changes in dopaminergic phenotype can be achieved.

Crosstalk between endoplasmic reticulum stress and brain inflammation in Alzheimer's disease

ABSTRACT While most often noted for its cognitive symptoms, Alzheimers disease (AD) is, at its core, a disease of protein misfolding/aggregation, with an intriguing inflammatory component. Defective clearance and/or abnormal production of the amyloid‐&bgr; peptide (A&bgr;), and its ensuing accumulation and aggregation, underlie two hallmark features of AD: brain accumulation of insoluble protein deposits known as amyloid or senile plaques, and buildup of soluble A&bgr; oligomers (A&bgr;Os), diffusible toxins linked to synapse dysfunction and memory impairment. In neurons, as in typical eukaryotic cells, the endoplasmic reticulum (ER) serves as a main compartment for the folding, maturation, trafficking and quality control of newly synthesized proteins. The ER lumen, a calcium‐rich, oxidizing environment, provides favorable conditions for these physiological functions to occur. These conditions, however, also favor protein aggregation. Several stressors, including metabolic/nutrient stress and certain pathologies, may upset the ER homeostasis, e.g., by affecting calcium levels or by causing the accumulation of unfolded or misfolded proteins. Whatever the underlying cause, the result is what is commonly known as “ER stress”. This, in turn, triggers a conserved cellular response mechanism known as the “unfolded protein response” (UPR). The UPR comprises three pathways involving transcriptional or translational regulators aimed at normalizing ER function, and each of them results in pro‐inflammatory signaling. A positive feedback loop exists between ER stress and inflammation, with clear implications for neurodegeneration and AD. Here, we explore recent findings on the role of ER stress and the UPR in inflammatory processes leading to synapse failure and memory impairment in AD. This article is part of the Special Issue entitled ‘Metabolic Impairment as Risk Factors for Neurodegenerative Disorders.’

Reply to Altered Monoaminergic Systems and Depressive-like Behavior in Congenic Prion Protein Knock-out Mice.

Nuvolone and Aguzzi (1) ascribed to the effect of a 129-Sirpa polymorphism, a macrophage phenotype we had previously attributed to PrP, and we acknowledge that comparisons of wild-type and Prnp-null mice are subject to the flanking gene problem, as are many other studies of genetically modified mice. However, our current suggestion of a PrPmonoaminergic link, consistent with the scaffold hypothesis, additionally relies on the binding of PrP to monoaminergic markers.