Sangeeta Nath

Spreading of Neurodegenerative Pathology via Neuron-to-Neuron Transmission of β-Amyloid

Alzheimers disease (AD) is the major cause of dementia. During the development of AD, neurofibrillary tangles progress in a fixed pattern, starting in the transentorhinal cortex followed by the hippocampus and cortical areas. In contrast, the deposition of β-amyloid (Aβ) plaques, which are the other histological hallmark of AD, does not follow the same strict spatiotemporal pattern, and it correlates poorly with cognitive decline. Instead, soluble Aβ oligomers have received increasing attention as probable inducers of pathogenesis. In this study, we use microinjections into electrophysiologically defined primary hippocampal rat neurons to demonstrate the direct neuron-to-neuron transfer of soluble oligomeric Aβ. Additional studies conducted in a human donor–acceptor cell model show that this Aβ transfer depends on direct cellular connections. As the transferred oligomers accumulate, acceptor cells gradually show beading of tubulin, a sign of neurite damage, and gradual endosomal leakage, a sign of cytotoxicity. These observations support that intracellular Aβ oligomers play a role in neurodegeneration, and they explain the manner in which Aβ can drive disease progression, even if the extracellular plaque load is poorly correlated with the degree of cognitive decline. Understanding this phenomenon sheds light on the pathophysiological mechanism of AD progression. Additional elucidation will help uncover the detailed mechanisms responsible for the manner in which AD progresses via anatomical connections and will facilitate the development of new strategies for stopping the progression of this incapacitating disease.

Spreading of Amyloid-β Peptides via Neuritic Cell-to-cell Transfer Is Dependent on Insufficient Cellular Clearance

The spreading of pathology through neuronal pathways is likely to be the cause of the progressive cognitive loss observed in Alzheimers disease (AD) and other neurodegenerative diseases. We have recently shown the propagation of AD pathology via cell-to-cell transfer of oligomeric amyloid beta (Aβ) residues 1-42 (oAβ1-42) using our donor-acceptor 3-D co-culture model. We now show that different Aβ-isoforms (fluorescently labeled 1-42, 3(pE)-40, 1-40 and 11-42 oligomers) can transfer from one cell to another. Thus, transfer is not restricted to a specific Aβ-isoform. Although different Aβ isoforms can transfer, differences in the capacity to clear and/or degrade these aggregated isoforms result in vast differences in the net amounts ending up in the receiving cells and the net remaining Aβ can cause seeding and pathology in the receiving cells. This insufficient clearance and/or degradation by cells creates sizable intracellular accumulations of the aggregation-prone Aβ1-42 isoform, which further promotes cell-to-cell transfer; thus, oAβ1-42 is a potentially toxic isoform. Furthermore, cell-to-cell transfer is shown to be an early event that is seemingly independent of later appearances of cellular toxicity. This phenomenon could explain how seeds for the AD pathology could pass on to new brain areas and gradually induce AD pathology, even before the first cell starts to deteriorate, and how cell-to-cell transfer can act together with the factors that influence cellular clearance and/or degradation in the development of AD.

Proteasome inhibition induces stress kinase dependent transport deficits--implications for Alzheimer's disease.

Alzheimers disease (AD) is characterized by accumulation of two misfolded and aggregated proteins, β-amyloid and hyperphosphorylated tau. Both cellular systems responsible for clearance of misfolded and aggregated proteins, the lysosomal and the proteasomal, have been shown to be malfunctioning in the aged brain and more so in patients with neurodegenerative diseases, including AD. This malfunction could be contributing to β-amyloid and tau accumulation, eventually aggregating in plaques and tangles. We have investigated the impact of decreased proteasome activity on tau phosphorylation as well as on microtubule stability and transport. To do this, we used our recently developed neuronal model where human SH-SY5Y cells obtain neuronal morphology and function through differentiation. We found that exposure to low doses of the proteasome inhibitor MG-115 caused tau phosphorylation, microtubule destabilization and disturbed neuritic transport. Furthermore, reduced proteasome activity activated several proteins implicated in tau phosphorylation and AD pathology, including c-Jun N-terminal kinase, c-Jun and extracellular signal-regulated protein kinase (ERK) 1/2. Restoration of the microtubule transport was achieved by inhibiting ERK 1/2 activation, and simultaneous inhibition of both ERK 1/2 and c-Jun reversed the proteasome inhibition-induced tau phosphorylation. Taken together, this study suggests that a decrease in proteasome activity can, through activation of c-Jun and ERK 1/2, result in several events related to neurodegenerative diseases. Restoration of proteasome activity or modulation of ERK 1/2 and c-Jun function can open new treatment possibilities against neurodegenerative diseases such as AD.

Protective properties of lysozyme on β-amyloid pathology: implications for Alzheimer disease.

The hallmarks of Alzheimer disease are amyloid-β plaques and neurofibrillary tangles accompanied by signs of neuroinflammation. Lysozyme is a major player in the innate immune system and has recently been shown to prevent the aggregation of amyloid-β1-40 in vitro. In this study we found that patients with Alzheimer disease have increased lysozyme levels in the cerebrospinal fluid and lysozyme co-localized with amyloid-β in plaques. In Drosophila neuronal co-expression of lysozyme and amyloid-β1-42 reduced the formation of soluble and insoluble amyloid-β species, prolonged survival and improved the activity of amyloid-β1-42 transgenic flies. This suggests that lysozyme levels rise in Alzheimer disease as a compensatory response to amyloid-β increases and aggregation. In support of this, in vitro aggregation assays revealed that lysozyme associates with amyloid-β1-42 and alters its aggregation pathway to counteract the formation of toxic amyloid-β species. Overall, these studies establish a protective role for lysozyme against amyloid-β associated toxicities and identify increased lysozyme in patients with Alzheimer disease. Therefore, lysozyme has potential as a new biomarker as well as a therapeutic target for Alzheimer disease.

Neuron-to-Neuron Transmission of Neurodegenerative Pathology

One of the hallmarks of neurodegenerative dementia diseases is the progressive loss of mental functions and the ability to manage activities of daily life. This progression is caused by the spread of the disease to more and more brain areas via anatomical connections. The pathophysiological process responsible for this spread of disease has long been sought after. There has been an increased understanding that the driving force of these neurodegenerative diseases could be the small, soluble intraneuronal accumulations of neurodegenerative proteins rather than the large, extracellular accumulations. Recently we have shown that the mechanism of spread of Alzheimer’s disease most likely depends on the neuron-to-neuron spread of such soluble intraneuronal accumulations of β-amyloid through neuritic connections. Similar transmissions have been shown for several other neurodegenerative proteins but little is known about the cellular mechanisms and about any potential strategies that might stop this spread. Resolving these questions requires good cellular models. We have established a unique model of synaptic transmission between human neuronal-like cells, something that has previously been difficult to target. This opens the possibility of developing potential inhibitors of progression of these devastating diseases.

Impact of high cholesterol in a Parkinson’s disease model: Prevention of lysosomal leakage versus stimulation of α-synuclein aggregation

Parkinsons disease is characterized by accumulation of intraneuronal cytoplasmic inclusions, Lewy bodies, which mainly consist of aggregated α-synuclein. Controversies exist as to whether high blood cholesterol is a risk factor for the development of the disease and whether statin treatment could have a protective effect. Using a model system of BE(2)-M17 neuroblastoma cells treated with the neurotoxin 1-methyl-4-phenylpyridinium (MPP+), we found that MPP+-induced cell death was accompanied by cholesterol accumulation in a lysosomal-like pattern in pre-apoptotic cells. To study the effects of lysosomal cholesterol accumulation, we increased lysosomal cholesterol through pre-treatment with U18666A and found delayed leakage of lysosomal contents into the cytosol, which reduced cell death. This suggests that increased lysosomal cholesterol is a stress response mechanism to protect lysosomal membrane integrity in response to early apoptotic stress. However, high cholesterol also stimulated the accumulation of α-synuclein. Treatment with the cholesterol-lowering drug lovastatin reduced MPP+-induced cell death by inhibiting the production of reactive oxygen species, but did not prevent lysosomal cholesterol increase nor affect α-synuclein accumulation. Our study indicates a dual role of high cholesterol in Parkinsons disease, in which it acts both as a protector against lysosomal membrane permeabilization and as a stimulator of α-synuclein accumulation.

Beneficial effects of increased lysozyme levels in Alzheimer’s disease modelled in Drosophila melanogaster

Genetic polymorphisms of immune genes that associate with higher risk to develop Alzheimers disease (AD) have led to an increased research interest on the involvement of the immune system in AD pathogenesis. A link between amyloid pathology and immune gene expression was suggested in a genome‐wide gene expression study of transgenic amyloid mouse models. In this study, the gene expression of lysozyme, a major player in the innate immune system, was found to be increased in a comparable pattern as the amyloid pathology developed in transgenic mouse models of AD. A similar pattern was seen at protein levels of lysozyme in human AD brain and CSF, but this lysozyme pattern was not seen in a tau transgenic mouse model. Lysozyme was demonstrated to be beneficial for different Drosophila melanogaster models of AD. In flies that expressed Aβ1‐42 or AβPP together with BACE1 in the eyes, the rough eye phenotype indicative of toxicity was completely rescued by coexpression of lysozyme. In Drosophila flies bearing the Aβ1‐42 variant with the Arctic gene mutation, lysozyme increased the fly survival and decreased locomotor dysfunction dose dependently. An interaction between lysozyme and Aβ1‐42 in the Drosophila eye was discovered. We propose that the increased levels of lysozyme, seen in mouse models of AD and in human AD cases, were triggered by Aβ1‐42 and caused a beneficial effect by binding of lysozyme to toxic species of Aβ1‐42, which prevented these from exerting their toxic effects. These results emphasize the possibility of lysozyme as biomarker and therapeutic target for AD.

Direct neuron to neuron transmission of β-amyloid oligomers causes neurodegeneration

Background: Progressive accumulation of aggregates distributing along anatomical pathways are the hall marks of common non-infectious neurodegenerative diseases like Alzheimer’s, Parkinson’s, frontotemporal dementia and Huntington’s. Recent reports demonstrated neuron to neuron transmission of the known pathogens of above diseases such as tau, a-synuclein and huntingtin and superoxide dismutase-1, suggesting mechanisms similar to prion transfer. Aß aggregates spreads close to the site of injected brain extracts from Alzheimer’s disease patients into the brain of amyloid precursor protein (APP) transgenic mice. Aß reduces the spine number and plasticity of neighboring cells and neuronal dysfunction induced by Aß can progress trans-synaptically. However it has not been demonstrated that neuron to neuron transmission of Aß actually takes place. Speculation of Prion like transmission have been put forward but its existence has until now remained elusive. Methods:Here we demonstrate transmission of Aß between neuronal cells using a donor and acceptor cells co-culturing and microinjection method. We show that exogenous Aß42 oligomers after uptake by differentiated human neuronal cell-line are subsequently transferred to co-cultured neuronal cells. Transfer from a single, microinjected hippocampal rat neuron to connected neurons was also shown. Results: The data presented here shows that oligomeric Aß is transmitted from neuron to neuron through neuritic connections which subsequently but slowly affects cells cytotoxicity. Oligomer spreading is restricted to neighboring rat neuron when injected. Transmission is very specific and seems to involve endosomal-lysosomal pathway, and is not inhibited by blockers of suggested Aß binding receptors. Conclusions: The finding of neuron to neuron transfer of Aß sheds important light on the propagation of Alzheimer’s disease through anatomical connections, suggesting similarities with other common neurodegenerative diseases, also dependent on cell nonautonomous mechanisms. This opens new avenues for the search for novel treatment.