Neuropathological role of PI3K/Akt/mTOR axis in Down syndrome brain

Down syndrome (DS) is the most frequent genetic cause of intellectual disability characterized by the presence of three copies of chromosome 21 (Chr21). Individuals with DS have sufficient neuropathology for a diagnosis of Alzheimer's disease (AD) after the age of 40 years. The aim of our study is to gain new insights in the molecular mechanisms impaired in DS subjects that eventually lead to the development of dementia. We evaluate the PI3K/Akt/mTOR axis in the frontal cortex from DS cases (under the age of 40 years) and DS with AD neuropathology compared with age-matched controls (Young and Old). The PI3K/Akt/mTOR axis may control several key pathways involved in AD that, if aberrantly regulated, affect amyloid beta (Aβ) deposition and tau phosphorylation. Our results show a hyperactivation of PI3K/Akt/mTOR axis in individuals with DS, with and without AD pathology, in comparison with respective controls. The PI3K/Akt/mTOR deregulation results in decreased autophagy, inhibition of IRS1 and GSK3β activity. Moreover, our data suggest that aberrant activation of the PI3K/Akt/mTOR axis acts in parallel to RCAN1 in phosphorylating tau, in DS and DS/AD. In conclusion, this study provides insights into the neuropathological mechanisms that may be engaged during the development of AD in DS. We suggest that deregulation of this signaling cascade is already evident in young DS cases and persist in the presence of AD pathology. The impairment of the PI3K/Akt/mTOR axis in DS population might represent a key-contributing factor to the neurodegenerative process that culminates in Alzheimer-like dementia.     

Redox proteomics analysis of HNE-modified proteins in Down syndrome brain: clues for understanding the development of Alzheimer disease

Down syndrome (DS) is the most common genetic cause of intellectual disability, due to partial or complete triplication of chromosome 21. DS subjects are characterized by a number of abnormalities including premature aging and development of Alzheimer disease (AD) neuropathology after approximately 40 years of age. Several studies show that oxidative stress plays a crucial role in the development of neurodegeneration in the DS population. Increased lipid peroxidation is one of the main events causing redox imbalance within cells through the formation of toxic aldehydes that easily react with DNA, lipids, and proteins. In this study we used a redox proteomics approach to identify specific targets of 4-hydroxynonenal modifications in the frontal cortex from DS cases with and without AD pathology. We suggest that a group of identified proteins followed a specific pattern of oxidation in DS vs young controls, probably indicating characteristic features of the DS phenotype; a second group of identified proteins showed increased oxidation in DS/AD vs DS, thus possibly playing a role in the development of AD. The third group of comparison, DS/AD vs old controls, identified proteins that may be considered specific markers of AD pathology. All the identified proteins are involved in important biological functions including intracellular quality control systems, cytoskeleton network, energy metabolism, and antioxidant response. Our results demonstrate that oxidative damage is an early event in DS, as well as dysfunctions of protein-degradation systems and cellular protective pathways, suggesting that DS subjects are more vulnerable to oxidative damage accumulation that might contribute to AD development. Further, considering that the majority of proteins have been already demonstrated to be oxidized in AD brain, our results strongly support similarities with AD in DS.     

Association between frontal cortex oxidative damage and beta-amyloid as a function of age in Down syndrome

Down syndrome (DS) is the most common genetic cause of intellectual disability in children, and the number of adults with DS reaching old age is increasing. By the age of 40 years, virtually all people with DS have sufficient neuropathology for a postmortem diagnosis of Alzheimer disease (AD). Trisomy 21 in DS leads to an overexpression of many proteins, of which at least two are involved in oxidative stress and AD: superoxide dismutase 1 (SOD1) and amyloid precursor protein (APP). In this study, we tested the hypothesis that DS brains with neuropathological hallmarks of AD have more oxidative and nitrosative stress than those with DS but without significant AD pathology, as compared with similarly aged-matched non-DS controls. The frontal cortex was examined in 70 autopsy cases (n = 29 control and n = 41 DS). By ELISA, we quantified soluble and insoluble Aβ40 and Aβ42, as well as oligomers. Oxidative and nitrosative stress levels (protein carbonyls, 4-hydroxy-2-trans-nonenal (HNE)-bound proteins, and 3-nitrotyrosine) were measured by slot-blot. We found that soluble and insoluble amyloid beta peptide (Aβ) and oligomers increase as a function of age in DS frontal cortex. Of the oxidative stress markers, HNE-bound proteins were increased overall in DS. Protein carbonyls were correlated with Aβ40 levels. These results suggest that oxidative damage, but not nitrosative stress, may contribute to the onset and progression of AD pathogenesis in DS. Conceivably, treatment with antioxidants may provide a point of intervention to slow pathological alterations in DS.     

The cholesterol transporter ABCG1 modulates the subcellular distribution and proteolytic processing of beta-amyloid precursor protein

Although intracellular cholesterol levels are known to influence the proteolysis of beta-amyloid precursor protein (APP), the effect of specific genes that regulate cholesterol metabolism on APP processing remains poorly understood. The cholesterol transporter ABCG1 facilitates cholesterol efflux to HDL and is expressed in brain. Notably, the human ABCG1 gene maps to chromosome 21q22.3, and individuals with Down syndrome (DS) typically manifest with Alzheimer's disease (AD) neuropathology in their 30s. Here, we demonstrate that expression of ABCG1 enhances amyloid-beta protein (Abeta) production in transfected HEK cells in a manner that requires functional cholesterol transporter activity. ABCG1-expressing cells also exhibit increased secreted APP (sAPP)alpha and sAPPbeta secretion and display increased cell surface-associated APP. These results suggest that ABCG1 increases the availability of APP as a secretase substrate for both the amyloidogenic and nonamyloidogenic pathways. In vivo, ABCG1 mRNA levels are 2-fold more abundant in DS brain compared with age- and sex-matched normal controls. Finally, both Abeta and sAPPalpha levels are increased in DS cortex relative to normal controls. These findings suggest that altered cholesterol metabolism and APP trafficking mediated by ABCG1 may contribute to the accelerated onset of AD neuropathology in DS.     

Hippocampal expression of cell‐adhesion glycoprotein neuroplastin is altered in Alzheimer's disease

Cell‐adhesion glycoprotein neuroplastin (Np) is involved in the regulation of synaptic plasticity and balancing hippocampal excitatory/inhibitory inputs which aids in the process of associative memory formation and learning. Our recent findings show that neuroplastin expression in the adult human hippocampus is specifically associated with major hippocampal excitatory pathways and is related to neuronal calcium regulation. Here, we investigated the hippocampal expression of brain‐specific neuroplastin isoform (Np65), its relationship with amyloid and tau pathology in Alzheimer's disease (AD), and potential involvement of neuroplastin in tissue response during the disease progression. Np65 expression and localization was analysed in six human hippocampi with confirmed AD neuropathology, and six age‐/gender‐matched control hippocampi by imunohistochemistry. In AD cases with shorter disease duration, the Np65 immunoreactivity was significantly increased in the dentate gyrus (DG), Cornu Ammonis 2/3 (CA2/3), and subiculum, with the highest level of Np expression being located on the dendrites of granule cells and subicular pyramidal neurons. Changes in the expression of neuroplastin in AD hippocampal areas seem to be related to the progression of disease. Our study suggests that cell‐adhesion protein neuroplastin is involved in tissue reorganization and is a potential molecular marker of plasticity response in the early neurodegeneration process of AD.     

Evidence against the overexpression of APP in Down syndrome

Down syndrome (DS) is the most common genetic disorder with mental retardation and is caused by trisomy 21. By the age of 40 years, virtually all adults with DS have sufficient neuropathology for a diagnosis of Alzheimer's disease (AD), which is characterized by accumulation of amyloid-beta in senile plaques and formation of neurofibrillary tangles. Amyloid-beta derives from a longer precursor protein, APP, whose gene maps to chromosome 21. In DS, the early appearance of senile plaques is commonly associated with the presence of a third copy of the APP gene. Here we show DS brains and trisomic fibroblasts in which APP is not overexpressed, compared to euploid controls, challenging the notion that the widespread amyloid-beta deposits, consistently found in DS individuals, result from an extra copy of APP.     

Regulator of calcineurin 1 (RCAN1) facilitates neuronal apoptosis through caspase-3 activation

Individuals with Down syndrome (DS) will inevitably develop Alzheimer disease (AD) neuropathology sometime after middle age, which may be attributable to genes triplicated in individuals with DS. The characteristics of AD neuropathology include neuritic plaques, neurofibrillary tangles, and neuronal loss in various brain regions. The mechanism underlying neurodegeneration in AD and DS remains elusive. Regulator of calcineurin 1 (RCAN1) has been implicated in the pathogenesis of DS. Our data show that RCAN1 expression is elevated in the cortex of DS and AD patients. RCAN1 expression can be activated by the stress hormone dexamethasone. A functional glucocorticoid response element was identified in the RCAN1 isoform 1 (RCAN1-1) promoter region, which is able to mediate the up-regulation of RCAN1 expression. Here we show that overexpression of RCAN1-1 in primary neurons activates caspase-9 and caspase-3 and subsequently induces neuronal apoptosis. Furthermore, we found that the neurotoxicity of RCAN1-1 is inhibited by knock-out of caspase-3 in caspase-3(-/-) neurons. Our study provides a novel mechanism by which RCAN1 functions as a mediator of stress- and Aβ-induced neuronal death, and overexpression of RCAN1 due to an extra copy of the RCAN1 gene on chromosome 21 contributes to AD pathogenesis in DS.     

Tau PET Imaging for Staging of Alzheimer's Disease in Down Syndrome

Alzheimer's disease (AD) pathology and early‐onset dementia develop almost universally in Down syndrome (DS). AD is defined neuropathologically by the presence of extracellular plaques of aggregated amyloid β protein and intracellular neurofibrillary tangles (NFTs) of aggregated hyperphosphorylated tau protein. The development of radiolabeled positron emission tomography (PET) ligands for amyloid plaques and tau tangles enables the longitudinal assessment of the spatial pattern of their accumulation in relation to symptomatology. Recent work indicates that amyloid pathology develops 15–20 years before neurodegeneration and symptom onset in the sporadic and autosomal dominant forms of AD, while tau pathology correlates more closely with symptomatic stages evidenced by cognitive decline and dementia. Recent work on AD biomarkers in DS illustrates similarities between DS and sporadic AD. It may soon be possible to apply recently developed staging classifications to DS to obtain a more nuanced understanding of the development AD in DS and to provide more accurate diagnosis and prognosis in the clinic.     

Neuropathological correlates of amyloid PET imaging in Down syndrome

Down syndrome (DS) results in an overproduction of amyloid‐β (Aβ) peptide associated with early onset of Alzheimer's disease (AD). DS cases have Aβ deposits detectable histologically as young as 12–30 years of age, primarily in the form of diffuse plaques, the type of early amyloid pathology also seen at pre‐clinical (i.e., pathological aging) and prodromal stages of sporadic late onset AD. In DS subjects aged >40 years, levels of cortical Aβ deposition are similar to those observed in late onset AD and in addition to diffuse plaques involve cored plaques associated with dystrophic neurites (neuritic plaques), which are of neuropathological diagnostic significance in AD. The purpose of this review is to summarize and discuss findings from amyloid PET imaging studies of DS in reference to postmortem amyloid‐based neuropathology. PET neuroimaging applied to subjects with DS has the potential to (a) track the natural progression of brain pathology, including the earliest stages of amyloid accumulation, and (b) determine whether amyloid PET biomarkers predict the onset of dementia. In addition, the question that is still incompletely understood and relevant to both applications is the ability of amyloid PET to detect Aβ deposits in their earliest form.     

Down syndrome,Alzheimer disease,and cerebral amyloid angiopathy: The complex triangle of brain amyloidosis

Down syndrome (DS) is the main genetic cause of intellectual disability worldwide. The overexpression of the Amyloid Precursor Protein, present in chromosome 21, leads to β‐amyloid deposition that results in Alzheimer disease (AD) and, in most cases, also to cerebral amyloid angiopathy (CAA) neuropathology. People with DS invariably develop the neuropathological hallmarks of AD at the age of 40, and they are at an ultra high risk for suffering AD‐related cognitive impairment thereafter. In the general population, cerebrovascular disease is a significant contributor to AD‐related cognitive impairment, while in DS remains understudied. This review describes the current knowledge on cerebrovascular disease in DS and reviews the potential biomarkers that could be useful in the future studies, focusing on CAA. We also discuss available evidence on sporadic AD or other genetically determined forms of AD. We highlight the urgent need of large biomarker‐characterized cohorts, including neuropathological correlations, to study the exact contribution of CAA and related vascular factors that play a role in cognition and occur with aging, their characterization and interrelationships. DS represents a unique context in which to perform these studies as this population is relatively protected from some conventional vascular risk factors and they develop significant CAA, DS represents a particular atheroma‐free model to study AD‐related vascular pathologies. Only deepening on these underlying mechanisms, new preventive and therapeutic strategies could be designed to improve the quality of life of this population and their caregivers and lead to new avenues of treatment also in the general AD population.     

Role of Amyloid Precursor Protein (APP) and Its Derivatives in the Biology and Cell Fate Specification of Neural Stem Cells

Amyloid precursor protein (APP) is a member of the APP family of proteins, and different enzymatic processing leads to the production of several derivatives that are shown to have distinct biological functions. APP is involved in the pathology of Alzheimer’s disease (AD), the most common neurodegenerative disorder causing dementia. Furthermore, it is believed that individuals with Down syndrome (DS) have increased APP expression, due to an extra copy of chromosome 21 (Hsa21), that contains the gene for APP. Nevertheless, the physiological function of APP remains unclear. It is known that APP plays an important role in neural growth and maturation during brain development, possibly by influencing proliferation, cell fate specification and neurogenesis of neural stem cells (NSCs). Proteolytic cleavage of APP occurs mainly via two mutually exclusive pathways, the non-amyloidogenic pathway or the amyloidogenic pathway. Other alternative pathways (η-secretase, δ-secretase and meprin pathways) have also been described for the physiological processing of APP. The different metabolites generated from these pathways, including soluble APPα (sAPPα), soluble APPβ (sAPPβ), β-amyloid (Aβ) peptides and the APP intracellular domain (AICD), have different functions determined by their structural differences, equilibrium and concentration with respect to other fragments derived from APP. This review discusses recent observations regarding possible functions of APP and its proteolytic derivatives in the biology and phenotypic specification of NSCs. This can be important for a better understanding of the pathogenesis and the development of future therapeutic applications for AD and/or DS, diseases in which alterations in neurogenesis have been described.     

Altered metabolism of the amyloid beta precursor protein is associated with mitochondrial dysfunction in Down's syndrome

Most Down's syndrome (DS) patients develop Alzheimer's disease (AD) neuropathology. Astrocyte and neuronal cultures derived from fetal DS brain show alterations in the processing of amyloid beta precursor protein (AbetaPP), including increased levels of AbetaPP and C99, reduced levels of secreted AbetaPP (AbetaPPs) and C83, and intracellular accumulation of insoluble Abeta42. This pattern of AbetaPP processing is recapitulated in normal astrocytes by inhibition of mitochondrial metabolism, consistent with impaired mitochondrial function in DS astrocytes. Intracellular Abeta42 and reduced AbetaPPs are also detected in DS and AD brains. The survival of DS neurons is markedly increased by recombinant or astrocyte-produced AbetaPPs, suggesting that AbetaPPs may be a neuronal survival factor. Thus, mitochondrial dysfunction in DS may lead to intracellular deposition of Abeta42, reduced levels of AbetaPPs, and a chronic state of increased neuronal vulnerability.     

Human brain nucleoside diphosphate kinase activity is decreased in Alzheimer's disease and Down syndrome

In brain, nucleoside diphosphate kinase (NDPK) and its coding gene, nm23, have been implicated to modulate neuronal cell proliferation, differentiation, and neurite outgrowth. However, a role of NDPK in neurodegenerative diseases has not been reported yet. Using proteomics techniques, we evaluated the protein levels of NDPK-A in seven brain regions from patients with Alzheimer's disease (AD) and Down syndrome (DS) showing AD-like neuropathology. NDPK-A was significantly decreased in brain regions (frontal, occipital, and parietal cortices) of both disorders. Due to the limitation of brain samples, the activity of NDPK was measured in three brain regions (frontal cortex, temporal cortex, and cerebellum). The specific activity of NDPK was significantly decreased in AD (frontal cortex) and DS (frontal and temporal cortices). Since NDPK-B could also drive the activity of NDPK, protein expression levels of both NDPK-A and NDPK-B were studied in frontal cortex by Western blot analysis. NDPK-A was significantly decreased in AD, which was consistent with the results of proteomics. However, NDPK-A was slightly decreased in DS and protein expression levels of NDPK-B in both DS and AD were moderately decreased, without reaching statistical significance. We propose that oxidative modification of NDPK could lead to the decreased activity of NDPK and, subsequently, influence several neuronal functions in neurodegenerative diseases as multifunctional enzyme through several mechanisms.     

Dual-specificity tyrosine(Y)-phosphorylation regulated kinase 1A-mediated phosphorylation of amyloid precursor protein: evidence for a functional link between Down syndrome and Alzheimer's disease

Most individuals with Down Syndrome (DS) show an early-onset of Alzheimer's disease (AD), which potentially results from the presence of an extra copy of a segment of chromosome 21. Located on chromosome 21 are the genes that encode β-amyloid (Aβ) precursor protein ( APP ), a key protein involved in the pathogenesis of AD, and dual-specificity tyrosine(Y)-phosphorylation regulated kinase 1A ( DYRK1A ), a proline-directed protein kinase that plays a critical role in neurodevelopment. Here, we describe a potential mechanism for the regulation of AD pathology in DS brains by DYRK1A-mediated phosphorylation of APP. We show that APP is phosphorylated at Thr668 by DYRK1A in vitro and in mammalian cells. The amounts of phospho-APP and Aβ are increased in the brains of transgenic mice that over-express the human DYRK1A protein. Furthermore, we show that the amounts of phospho-APP as well as those of APP and DYRK1A are elevated in human DS brains. Taken together, these results reveal a potential regulatory link between APP and DYRK1A in DS brains, and suggest that the over-expression of DYRK1A in DS may play a role in accelerating AD pathogenesis through phosphorylation of APP.     

The zinc dyshomeostasis hypothesis of Alzheimer's disease

Alzheimer's disease (AD) is the most common form of dementia in the elderly. Hallmark AD neuropathology includes extracellular amyloid plaques composed largely of the amyloid-β protein (Aβ), intracellular neurofibrillary tangles (NFTs) composed of hyper-phosphorylated microtubule-associated protein tau (MAP-tau), and microtubule destabilization. Early-onset autosomal dominant AD genes are associated with excessive Aβ accumulation, however cognitive impairment best correlates with NFTs and disrupted microtubules. The mechanisms linking Aβ and NFT pathologies in AD are unknown. Here, we propose that sequestration of zinc by Aβ-amyloid deposits (Aβ oligomers and plaques) not only drives Aβ aggregation, but also disrupts zinc homeostasis in zinc-enriched brain regions important for memory and vulnerable to AD pathology, resulting in intra-neuronal zinc levels, which are either too low, or excessively high. To evaluate this hypothesis, we 1) used molecular modeling of zinc binding to the microtubule component protein tubulin, identifying specific, high-affinity zinc binding sites that influence side-to-side tubulin interaction, the sensitive link in microtubule polymerization and stability. We also 2) performed kinetic modeling showing zinc distribution in extra-neuronal Aβ deposits can reduce intra-neuronal zinc binding to microtubules, destabilizing microtubules. Finally, we 3) used metallomic imaging mass spectrometry (MIMS) to show anatomically-localized and age-dependent zinc dyshomeostasis in specific brain regions of Tg2576 transgenic, mice, a model for AD. We found excess zinc in brain regions associated with memory processing and NFT pathology. Overall, we present a theoretical framework and support for a new theory of AD linking extra-neuronal Aβ amyloid to intra-neuronal NFTs and cognitive dysfunction. The connection, we propose, is based on β-amyloid-induced alterations in zinc ion concentration inside neurons affecting stability of polymerized microtubules, their binding to MAP-tau, and molecular dynamics involved in cognition. Further, our theory supports novel AD therapeutic strategies targeting intra-neuronal zinc homeostasis and microtubule dynamics to prevent neurodegeneration and cognitive decline.     

CASP3 protein expression by flow cytometry in Down’s syndrome subjects

Down’s syndrome (DS), the most common chromosomal disorder, is caused by 21 trisomy and is featured by intellectual disability. Subjects with DS can develop some traits of Alzheimer disease (AD) at an earlier age than subjects without trisomy 21. Apoptosis is a programmed cell death process under both normal physiological and pathological conditions. Caspase-3 (CASP3) plays an important role in neuronal death during nervous system development and under certain pathological conditions. Furthermore, in vitro and in vivo studies report elevated expression and activation of CASP3 in models of AD. On this account, the expression of CASP3 gene was evaluated in cultures of fibroblasts of DS and normal subjects by flow cytometry. CASP3 protein was up-regulated in fibroblasts of DS. The data obtained from this study strengthen the hypothesis that the over-expression of CASP3 gene could have a role in the activation of the apoptotic pathways acting in the neurodegenerative processes in DS.     

Potential neuroprotective strategies against tauopathy

Tauopathies are neurodegenerative diseases, including AD (Alzheimer's disease) and FTLD-T (tau-positive frontotemporal lobar degeneration), with shared pathology presenting as accumulation of detergent-insoluble hyperphosphorylated tau deposits in the central nervous system. The currently available treatments for AD address only some of the symptoms, and do not significantly alter the progression of the disease, namely the development of protein aggregates and loss of functional neurons. The development of effective treatments for various tauopathies will require the identification of common mechanisms of tau neurotoxicity, and pathways that can be modulated to protect against neurodegeneration. Model organisms, such as Caenorhabditis elegans, provide methods for identifying novel genes and pathways that are involved in tau pathology and may be exploited for treatment of various tauopathies. In the present paper, we summarize data regarding characterization of MSUT2 (mammalian suppressor of tau pathology 2), a protein identified in a C. elegans tauopathy model and subsequently shown to modify tau toxicity in mammalian cell culture via the effects on autophagy pathways. MSUT2 represents a potential drug target for prevention of tau-related neurodegeneration.     

Protein expression in Down syndrome brain

Down syndrome (DS) is the most common chromosomal abnormality associated with early mental retardation and neurological abnormalities followed by precocious age dependent Alzheimer-type neurode generation later in life. Knowledge of the pathological mechanisms involved in DS is far from complete, but overexpression of genes residing in chromosome 21 was considered to be the central point for the DS phenotype. In this regard, beta amyloid precursor protein (APP), CuZn superoxide dismutase (SOD1) and S100beta have been implicated in causing apoptosis, a mechanism thought to be responsible for neuronal loss in DS, in one way or another. The gene dosage hypothesis has been challenged, however, and dysregulation of expression of genes located on other chromosomes has been described, which may well be secondary to chromosomal imbalance or a direct consequence of the disease process. The present review focuses on the protein expression profile in DS and we postulate that abnormalities in the coordinated expression, as well as interaction of proteins may be responsible for the neuropathology of DS. A series of candidate proteins are discussed that may be directly causing or reflecting the DS phenotype, in particular the brain abnormalities in DS.     

A role for thrombospondin-1 deficits in astrocyte-mediated spine and synaptic pathology in Down's syndrome

Background

Down''s syndrome (DS) is the most common genetic cause of mental retardation. Reduced number and aberrant architecture of dendritic spines are common features of DS neuropathology. However, the mechanisms involved in DS spine alterations are not known. In addition to a relevant role in synapse formation and maintenance, astrocytes can regulate spine dynamics by releasing soluble factors or by physical contact with neurons. We have previously shown impaired mitochondrial function in DS astrocytes leading to metabolic alterations in protein processing and secretion. In this study, we investigated whether deficits in astrocyte function contribute to DS spine pathology.

Methodology/Principal Findings

Using a human astrocyte/rat hippocampal neuron coculture, we found that DS astrocytes are directly involved in the development of spine malformations and reduced synaptic density. We also show that thrombospondin 1 (TSP-1), an astrocyte-secreted protein, possesses a potent modulatory effect on spine number and morphology, and that both DS brains and DS astrocytes exhibit marked deficits in TSP-1 protein expression. Depletion of TSP-1 from normal astrocytes resulted in dramatic changes in spine morphology, while restoration of TSP-1 levels prevented DS astrocyte-mediated spine and synaptic alterations. Astrocyte cultures derived from TSP-1 KO mice exhibited similar deficits to support spine formation and structure than DS astrocytes.

Conclusions/Significance

These results indicate that human astrocytes promote spine and synapse formation, identify astrocyte dysfunction as a significant factor of spine and synaptic pathology in the DS brain, and provide a mechanistic rationale for the exploration of TSP-1-based therapies to treat spine and synaptic pathology in DS and other neurological conditions.