Zebrafish as a tool in Alzheimer's disease research

Alzheimer's disease is the most prevalent form of neurodegenerative disease. Despite many years of intensive research our understanding of the molecular events leading to this pathology is far from complete. No effective treatments have been defined and questions surround the validity and utility of existing animal models. The zebrafish (and, in particular, its embryos) is a malleable and accessible model possessing a vertebrate neural structure and genome. Zebrafish genes orthologous to those mutated in human familial Alzheimer's disease have been defined. Work in zebrafish has permitted discovery of unique characteristics of these genes that would have been difficult to observe with other models. In this brief review we give an overview of Alzheimer's disease and transgenic animal models before examining the current contribution of zebrafish to this research area. This article is part of a Special Issue entitled Zebrafish Models of Neurological Diseases.     

An N-terminal fragment of ProSAAS (a granin-like neuroendocrine peptide precursor) is associated with tau inclusions in Pick's disease

The deposition of aggregated tau in cytoplasmic inclusions is one of the common neuropathological features in various dementing neurodegenerative disorders. At present, it remains unclear whether tau inclusions exert neurotoxicity or they are simply the consequence of neurodegeneration. In our approach for the analysis of the composition of tau inclusions, we detected the intense binding of anti-diacylglycerol kinase-zeta (DGK-zeta) antibodies to Pick bodies (PBs), which represent tau inclusions in Pick's disease. The polyclonal antibodies were found to cross-react with a 21-kDa protein, but not with tau or ubiquitin, on Western blots of normal human brain extracts. Analysis of the 21-kDa protein by two-dimensional-gel electrophoresis and mass-spectrometry revealed that the protein is an N-terminal fragment of proSAAS (a human granin-like neuroendocrine peptide precursor). Our results suggest that sequestration of the N-terminal fragment of proSAAS in intracellular PBs may cause a functional disturbance of neurons in Pick's disease.     

Ceruloplasmin immunoreactivity in neurodegenerative disorders

Ceruloplasmin (CP) is a 132kd cuproprotein which, together with transferrin, provides the majority of anti-oxidant capacity in serum. Increased iron deposition and lipid peroxidation in the basal ganglia of subjects with hereditary CP deficiency suggest that CP may serve as an anti-oxidant in the brain as well. The present study compared CP immunoreactivity in brain specimens from normal controls and subjects with neurodegenerative disorders (Alzheimer's disease [AD], Parkinson's disease [PD], progressive supranuclear palsy [PSP], and Huntington's disease [HD]) (n = 5 per group). The relative intensity of neuronal CP staining and the numbers of CP-stained neurons per 25x microscope field were determined in hippocampus (CA1, subiculum, and parahippocampal gyrus), parietal cortex, frontal cortex, substantia nigra, and caudate. CP was detected in both neurons and astrocytes in all specimens, and in senile plaques and occasional neurofibrillary tangles in AD brain. Neuronal CP staining intensity tended to increase in most AD brain regions, but was statistically significant vs controls only in the CA1 region of hippocampus (p = .016). Neuronal CP staining in brain specimens from other neurodegenerative disorders showed a slight but nonsignificant increase vs controls. The numbers of CP-stained neurons per field did not differ between the various neurodegenerative disorders and controls. These results suggest that a modest increase in neuronal CP content is present in the AD brain, and lesser elevations in neuronal CP occur in the other neurodegenerative disorders in this study. Though CP functions as both an acute phase protein and an anti-oxidant in peripheral tissues, whether it does so in the brain remains to be determined.     

Neurofibrillary degeneration in progressive supranuclear palsy and corticobasal degeneration: tau pathologies with exclusively "exon 10" isoforms

Pathological tau proteins that constitute the basic matrix of neuronal inclusions observed in numerous neurodegenerative disorders are disease specific. This is mainly the consequence of the aggregation of specific sets of tau isoforms according to the diseases, i.e., six isoforms in Alzheimer's disease (AD) and exclusively the three tau isoforms lacking the corresponding sequence of exon 10 (E10-) in Pick's disease (PiD). By using antibodies specific to the different tau isoforms and one- and two-dimensional gel electrophoresis followed by western blots, we demonstrate herein a third group of neurodegenerative disorders characterized by intraneuronal inclusions exclusively constituted of tau isoforms containing the sequence corresponding to exon 10, progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD). Together, tau isoforms with exon 10 clearly differentiate three groups of neurodegenerative diseases: AD, PiD, and PSP/CBD. For each group, the neuropathological and clinical phenotypes are most likely related to specific sets of tau isoforms expressed by the vulnerable neuronal populations. The recently described mutations of the tau gene responsible for familial frontotemporal dementias also support this hypothesis.     

The complex relationship between soluble and insoluble tau in tauopathies revealed by efficient dephosphorylation and specific antibodies

Phosphorylated tau is deposited as insoluble inclusion bodies in the tauopathies. We have used a new efficient method to dephosphorylate tau extracted from control and tauopathy brain. In some tauopathies, including Alzheimer's disease and progressive supranuclear palsy, the pattern of insoluble tau isoforms reflected that of soluble tau. In contrast, in corticobasal degeneration, Pick's disease, and some forms of fronto-temporal dementia, specific tau isoforms were selectively sequestered into insoluble inclusion-forming tau. Therefore the overall expression of individual tau isoforms does not predict which tau isoforms are deposited in all tauopathies and different mechanisms must operate that result in the deposition of specific tau isoforms.     

Regulation of autophagy by polyphenols: Paving the road for treatment of neurodegeneration

In the present paper, we will discuss on the importance of autophagy in the central nervous system, and outline the relation between autophagic pathways and the pathogenesis of neurodegenerative disorders. The potential therapeutic benefits of naturally occurring phytochemicals as pharmacological modulators of autophagy will also be addressed. Our findings provide renewed insight on the molecular modes of protection by polyphenols, which is likely to be at least in part mediated not only by their potent antioxidant and anti-inflammatory effects, but also through modulation of autophagic processes to remove the aberrant protein aggregates.     

Proteomic and functional alterations in brain mitochondria from Tg2576 mice occur before amyloid plaque deposition

Synaptic dysfunction is an early event in Alzheimer's disease patients and has also been detected in transgenic mouse models. In the present study, we analyzed proteomic changes in synaptosomal fractions from Tg2576 mice that overexpress mutant human amyloid precursor protein (K670N, M671L) and from their nontransgenic littermates. Cortical and hippocampal tissue was microdissected at the onset of cognitive impairment, but before deposition of amyloid plaques. Crude synaptosomal fractions were prepared by differential centrifugation, proteins were separated by 2-D DIGE and identified by MS/MS. Significant alterations were detected in mitochondrial heat shock protein 70 pointing to a mitochondrial stress response. Subsequently, synaptosomal versus nonsynaptic mitochondria were purified from Tg2576 mice brains by density gradient centrifugation. Mitochondrial proteins were separated by IEF or Blue-native gel electrophoresis in the first dimension and SDS-PAGE in the second dimension. Numerous changes in the protein subunit composition of the respiratory chain complexes I and III were identified. Levels of corresponding mRNAs remain unchanged as shown by Affymetrix oligonucleotide array analysis. Functional examination revealed impaired state 3 respiration and uncoupled respiration in brain mitochondria from young Tg2576 mice. By immunoblotting, amyloid-beta oligomers were detected in synaptosomal fractions from Tg2576 mice and reduced glucose metabolism was observed in Tg2576 mice brains by [14C]-2-deoxyglucose infusion. Taken together, we demonstrate alterations in the mitochondrial proteome and function that occur in Tg2576 mice brains before amyloid plaque deposition suggesting that mitochondria are early targets of amyloid-beta aggregates.     

Age-related changes of brain iron load changes in the frontal cortex in APPswe/PS1ΔE9 transgenic mouse model of Alzheimer's disease

Alzheimer's disease (AD) as a neurodegenerative brain disorder is a devastating pathology leading to disastrous cognitive impairments and dementia, associated with major social and economic costs to society. Iron can catalyze damaging free radical reactions. With age, iron accumulates in brain frontal cortex regions and may contribute to the risk of AD. In this communication, we investigated the age-related brain iron load changes in the frontal cortex of 6- and 12-month-old C57BL/6J (C57) and APP_swe/PS1_ΔE9 (APP/PS1) double transgenic mouse by using graphite furnace atomic absorption spectrometry (GFAAS) and Perls’ reaction. In the present study, we also evaluated the age-related changes of DMT1 and FPN1 by using Western blot and qPCR. We found that compared with 6-month-old APP/PS1 mice and the 12-month-old C57 mice, the 12-month-old APP/PS1 mice had increased iron load in the frontal cortex. The levels of DMT1 were significantly increased and the FPN1 were significantly reduced in the frontal cortex of the 12-month-old APP/PS1 mice than that in the 6-month-old APP/PS1 mice and 12-month-old C57 mice. We conclude that in AD damage occurs in conjunction with iron accumulation, and the brain iron load associated with loss control of the brain iron metabolism related protein DMT1 and FPN1 expressions.     

Understanding the importance of autophagy in human diseases using Drosophila

Autophagy is a lysosome-dependent intracellular degradation pathway that has been implicated in the pathogenesis of various human diseases, either positively or negatively impacting disease outcomes depending on the specific context. The majority of medical conditions including cancer, neurodegenerative diseases, infections and immune system disorders and inflammatory bowel disease could probably benefit from therapeutic modulation of the autophagy machinery. Drosophila represents an excellent model animal to study disease mechanisms thanks to its sophisticated genetic toolkit, and the conservation of human disease genes and autophagic processes. Here, we provide an overview of the various autophagy pathways observed both in flies and human cells(macroautophagy, microautophagy and chaperone-mediated autophagy), and discuss Drosophila models of the above-mentioned diseases where fly research has already helped to understand how defects in autophagy genes and pathways contribute to the relevant pathomechanisms.     

Induction of clusterin Expression by Neuronal Cell Death in Zebrafish

Clusterin, a protein associated with multiple functions, is expressed in a wide variety of mammalian tissues. Although clusterin is known to be involved in neurodegenerative diseases, ageing, and tumorigenesis, a detailed analysis of the consequences of gain- or loss-of-function approaches has yet to be performed to understand the underlying mechanisms of clusterin functions. Since clusterin levels change in neurological diseases, it is likely that clusterin contributes to cell death and degeneration in general. Zebrafish was investigated as a model system to study human diseases. During development, zebrafish clusterin was expressed in the notochord and nervous system. Embryonic overexpression of clusterin by mRNA microinjection did not affect axis formation, whereas its knock-down by anti-sense morpholino treatment resulted in neuronal cell death. To analyze the function of clusterin in neurodegeneration, a transgenic zebrafish was investigated, in which nitroreductase expression is regulated under the control of a neuron-specific huC promoter which is active between the stages of early neuronal precursors and mature neurons. Nitroreductase turns metronidazole into a cytotoxic agent that induces cell death within 12 h. After metronidazole treatment, transgenic zebrafish showed neuron-specific cell death. Interestingly, we also observed a dramatic induction of clusterin expression in the brain and spinal cord in these fish, suggesting a direct or indirect role of clusterin in neuronal cell death and thus, more generally, in neurodegeneration.     

The role of Sirt1: At the crossroad between promotion of longevity and protection against Alzheimer's disease neuropathology

Sirt1, a mammalian member of the sirtuin gene family, holds great potential for promoting longevity, preventing against disease and increasing cell survival. For example, studies suggest that the beneficial impact of caloric restriction in promoting longevity and cellular function may be mediated, in part, by Sirt1 through mechanisms involving PGC-1α, which plays important role in the regulation of cellular metabolism and inflammatory and antioxidant responses. Sirt1 may also interfere with mechanisms implicated in pathological disorders. We will present recent evidence indicating that Sirt1 may protect against Alzheimer's disease by interfering with the generation of β-amyloid peptides. We will discuss Sirt1 as a potential novel target, in addition to the development of Sirt1 activators for the prevention and treatment of Alzheimer's disease.     

Exploration of multi‐target effects of 3‐benzoyl‐5‐hydroxychromen‐2‐one in Alzheimer’s disease cell and mouse models

Microtubule‐associated protein Tau, abundant in the central nervous system (CNS), plays crucial roles in microtubule assembly and stabilization. Abnormal Tau phosphorylation and aggregation are a common pathogenic hallmark in Alzheimer's disease (AD). Hyperphosphorylation of Tau could change its conformation and result in self‐aggregation, increased oxidative stress, and neuronal death. In this study, we examined the potential of licochalcone A (a natural chalcone) and five synthetic derivatives (LM compounds) for inhibiting Tau misfolding, scavenging reactive oxygen species (ROS) and providing neuroprotection in human cells expressing proaggregant ΔK280 Tau_RD‐DsRed. All test compounds were soluble up to 100 μM in cell culture media and predicted to be orally bioavailable and CNS‐active. Among them, licochalcone A and LM‐031 markedly reduced Tau misfolding and associated ROS, promoted neurite outgrowth, and inhibited caspase 3 activity in ΔK280 Tau_RD‐DsRed 293 and SH‐SY5Y cells. Mechanistic studies showed that LM‐031 upregulates HSPB1 chaperone, NRF2/NQO1/GCLC pathway, and CREB‐dependent BDNF/AKT/ERK/BCL2 pathway in ΔK280 Tau_RD‐DsRed SH‐SY5Y cells. Decreased neurite outgrowth upon induction of ΔK280 Tau_RD‐DsRed was rescued by LM‐031, which was counteracted by knockdown of NRF2 or CREB. LM‐031 further rescued the downregulated NRF2 and pCREB, reduced Aβ and Tau levels in hippocampus and cortex, and ameliorated cognitive deficits in streptozocin‐induced hyperglycemic 3 × Tg‐AD mice. Our findings strongly indicate the potential of LM‐031 for modifying AD progression by targeting HSPB1 to reduce Tau misfolding and activating NRF2 and CREB pathways to suppress apoptosis and promote neuron survival, thereby offering a new drug development avenue for AD treatment.     

Bin1 antibody lowers the expression of phosphorylated Tau in Alzheimer's disease

Alzheimer's disease (AD) is an irreversible, progressive brain disorder responsible for memory loss leading to the inability to carry out the simplest tasks. AD is one of the leading causes of death in the United States. As yet there are no effective medications to treat this debilitating disease. In recent years, a human gene called bridging integrator 1 (BIN1) has emerged as one of the most important genes in affecting the incidence of sporadic AD. Bin1 can directly bind to Tau and mediates late onset AD risk by modulating Tau pathology. Recently our group found Bin1 antibody could exert drug-like properties in an animal model of ulcerative colitis. We hypothesized that the Bin1 monoclonal antibody (mAb) could be used in the treatment of AD by lowering the levels of Tau in cell culture and animal models. Cell culture studies confirmed that the Bin1 mAb (99D) could lower the levels of phosphorylated Tau (pTau). Multiple mechanisms aided by endosomal proteins and Fc gamma receptors are involved in the uptake of Bin1 mAb into cells. In Tau expressing cell culture, the Bin1 mAb induces the proteasome machinery leading to ubiquitination of molecules thereby preventing cell stress. In vivo studies demonstrated that treatment of P301S mice expressing Tau with the Bin1 mAb survived longer than the untreated mice. Our data confirm that Bin1 mAb lowers the levels of pTau and could be a drug candidate in the treatment of AD.     

星形胶质细胞在阿尔茨海默病中的功能变化及分子机制

星形胶质细胞在脑内数量最多,分布最广,对神经元有营养支持的作用,并且能够调控神经元的活性。越来越多的证据表明星形胶质细胞激活参与阿尔茨海默病(Alzheimer's disease,AD)的发生和发展。在AD病理情况下,星形胶质细胞在多种因子如β淀粉样蛋白(beta-amyloid,Aβ)和促炎细胞因子的作用下被激活,激活的星形胶质细胞进一步释放一氧化氮(Nitric oxide,NO)和多种炎性因子增强炎症级联反应。功能失常的星形胶质细胞会促进Aβ的产生,减弱对Aβ的摄取和清除,导致Aβ聚集沉积形成老年斑。激活的星形胶质细胞释放的炎症因子还能显著增加神经元内tau蛋白的异常过度磷酸化,产生神经纤维缠结。本文对星形胶质细胞在AD中参与神经变性的功能变化和分子机制进行总结,为星形胶质细胞作为靶点预防及治疗AD提供一定的理论依据。     

Densité des dépôts de la protéine Tau versus activité métabolique cortico-cérébrale chez des patients atteints de maladie d’Alzheimer ou de pathologies apparentées

IntroductionThe extracellular deposits of major senile plaques composed of Aβ proteins and intracellular degenerations or neurofibrillary degenerations (NFD) made up of hyperphosphorylated tau proteins are characteristic of Alzheimer's disease (AD). These characteristic lesions develop well before the first symptoms. NFD lesions seem to correlate with clinical symptomatology and allow Alzheimer's disease to be classified into neuropathological stages. In this study, we wanted to compare metabolic activity and NFD density by molecular imaging in a small cohort of subjects.Materials and methodsIn this study we evaluated the binding profile (brain density and distribution) of tau aggregates using [¹⁸F] – AV-1451 ([¹⁸F] -T807 or flortaucipir) PET in a group of 7 patients with clinical diagnosis of AD or related neurodegenerative pathology but with a very variable evolutionary profile (MMS between 15 and 25). All subjects also underwent a [¹⁸F] – FDG PET scan to assess their neuronal metabolic activity. For each subject, [¹⁸F] – T807 binding intensity and degree of hypometabolism were visually quantified in 5 levels for each predefined cortical region.ResultsFour subjects had a hypometabolic pattern supporting their diagnosis and which, moreover, was consistent with the distribution of NFD, however with slightly more extensive NFD lesions, particularly in the occipital region. One subject had neither hypometabolism nor a significant NFD array. One subject had predominantly hypometabolism in the frontotemporal cortex without significant lesion of DNF. The 7th subject presented diffuse cortical hypometabolism with few NFD lesions.ConclusionDespite a limited number of subjects, results seem to confirm the close link between the presence of the NFD lesions visible on tau PET imaging and neurodegeneration in AD.     

Amyloidogenesis of Tau protein

The role of microtubule‐associated protein Tau in neurodegeneration has been extensively investigated since the discovery of Tau amyloid aggregates in the brains of patients with Alzheimer's disease (AD). The process of formation of amyloid fibrils is known as amyloidogenesis and attracts much attention as a potential target in the prevention and treatment of neurodegenerative conditions linked to protein aggregation. Cerebral deposition of amyloid aggregates of Tau is observed not only in AD but also in numerous other tauopathies and prion diseases. Amyloidogenesis of intrinsically unstructured monomers of Tau can be triggered by mutations in the Tau gene, post‐translational modifications, or interactions with polyanionic molecules and aggregation‐prone proteins/peptides. The self‐assembly of amyloid fibrils of Tau shares a number of characteristic features with amyloidogenesis of other proteins involved in neurodegenerative diseases. For example, in vitro experiments have demonstrated that the nucleation phase, which is the rate‐limiting stage of Tau amyloidogenesis, is shortened in the presence of fragmented preformed Tau fibrils acting as aggregation templates (“seeds”). Accordingly, Tau aggregates released by tauopathy‐affected neurons can spread the neurodegenerative process in the brain through a prion‐like mechanism, originally described for the pathogenic form of prion protein. Moreover, Tau has been shown to form amyloid strains—structurally diverse self‐propagating aggregates of potentially various pathological effects, resembling in this respect prion strains. Here, we review the current literature on Tau aggregation and discuss mechanisms of propagation of Tau amyloid in the light of the prion‐like paradigm.     

Tau 蛋白磷酸化在阿尔茨海默病中所处的地位

阿尔茨海默病(Alzheimer’s disease,AD)是一种老年人常见的神经系统退行性疾病,是痴呆最常见的病因。AD患者越来越多,给家属及社会带来严重负担造成了巨大的家庭和社会负担,这就迫使我们进一步探讨AD发病机制。在AD的众多发病机制中,tau蛋白假说倍受青睐。在蛋白磷酸酯酶2A(protein phosphatase 2A,PP2A)、糖原合酶激酶-3β(glycogen synthase kinase-3β,GSK-3β)、细胞周期依赖性蛋白激酶-5(cyclin-dependent kinase 5,CDK-5)和Bcl-2等蛋白酶及调节蛋白作用下,微管相关蛋白tau蛋白以其异常磷酸化结构或是形成二聚体、寡聚体和神经原纤维缠结等形式,参与到AD的病理过程。Tau蛋白及其相关结构,可能启动或促进了AD的凋亡,亦可能抑制了急性凋亡却促进了慢性的神经细胞变性。揭开这一谜底,可能揭开AD病理改变的神秘面纱。