[Show abstract][Hide abstract] ABSTRACT: Aβ oligomers (AβOs) are crucially involved in Alzheimer's Disease (AD). However, the lack of selective approaches for targeting these polymorphic Aβ assemblies represents a major hurdle in understanding their biosynthesis, traffic and actions in living cells. Here, we established a subcellularly localized conformational-selective interference (CSI) approach, based on the expression of a recombinant antibody fragment against AβOs in the endoplasmic reticulum (ER). By CSI, we can control extra- and intracellular pools of AβOs produced in an AD-relevant cell model, without interfering with the maturation and processing of the Aβ precursor protein. The anti-AβOs intrabody selectively intercepts critical AβO conformers in the ER, modulating their assembly and controlling their actions in pathways of cellular homeostasis and synaptic signalling. Our results demonstrate that intracellular Aβ undergoes pathological oligomerization through critical conformations formed inside the ER. This establishes intracellular AβOs as key targets for AD treatment and presents CSI as a potential targeting strategy.
[Show abstract][Hide abstract] ABSTRACT: The 7WD4 and 7PA2 cell lines, widely used as cellular models for Alzheimer's disease (AD), have been used to investigate the effects of amyloid-β protein precursor overexpression and amyloid-β (Aβ) oligomer accumulation on mitochondrial function. Under standard culture conditions, both cell lines, compared to Chinese hamster ovary (CHO) control cells, displayed an ~5% decrease of O2 respiration as sustained by endogenous substrates. Functional impairment of the respiratory chain was found distributed among the protein complexes, though more evident at the level of complex I and complex IV. Measurements of ATP showed that its synthesis by oxidative phosphorylation is decreased in 7WD4 and 7PA2 cells by ~25%, this loss being partly compensated by glycolysis (Warburg effect). Compensation proved to be more efficient in 7WD4 than in 7PA2 cells, the latter cell line displaying the highest reactive oxygen species production. The strongest deficit was observed in mitochondrial membrane potential that is almost 40% and 60% lower in 7WD4 and 7PA2 cells, respectively, in comparison to CHO controls. All functional parameters point to a severe bioenergetic impairment of the AD cells, with the extent of mitochondrial dysfunction being correlated to the accumulation of Aβ peptides and oligomers.
[Show abstract][Hide abstract] ABSTRACT: Several open questions call for new studies on pathogenic mechanisms leading to Alzheimers Disease (AD), with the search for upstream drivers of the neurodegeneration cascade, such as neurotrophic deficits, early misfolding events of AD-related proteins (Abeta and tau) and understanding the multifactorial basis of AD pathogenesis. Since seminal immunosympathectomy experiment which represents the first example of a knock out experiment (albeit a protein knock-out), antibodies have had a long and successful history as a tool to selectively interfere with the function of proteins in cells and in organisms and antibody technologies represent a major weapon in the set of target validation techniques. Here, we describe a technology, pioneered by our group, based on recombinant antibody domains exploited as intracellular antibodies (intrabodies) whereby antibodies are used as genes, rather than as proteins. We discuss several applications and new promising developments of the intrabody approach for protein interference, especially in the field of AD research.
Journal of biological regulators and homeostatic agents 04/2013; 27(2 Suppl):89-105. · 2.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: ProNGF, the precursor of mature nerve growth factor (NGF), is the most abundant form of NGF in the brain. ProNGF and mature NGF differ significantly in their receptor interaction properties and in their bioactivity. ProNGF increases markedly in the cortex of Alzheimer's disease (AD) brains and proNGF\NGF imbalance has been postulated to play a role in neurodegeneration. However, a direct proof for a causal link between increased proNGF and AD neurodegeneration is lacking. In order to evaluate the consequences of increased levels of proNGF in the postnatal brain, transgenic mice expressing a furin cleavage-resistant form of proNGF, under the control of the neuron-specific mouse Thy1.2 promoter, were derived and characterized. Different transgenic lines displayed a phenotypic gradient of neurodegenerative severity features. We focused the analysis on the two lines TgproNGF#3 and TgproNGF#72, which shared learning and memory impairments in behavioral tests, cholinergic deficit and increased Aβ-peptide immunoreactivity. In addition, TgproNGF#3 mice developed Aβ oligomer immunoreactivity, as well as late diffuse astrocytosis. Both TgproNGF lines also display electrophysiological alterations related to spontaneous epileptic-like events. The results provide direct evidence that alterations in the proNGF/NGF balance in the adult brain can be an upstream driver of neurodegeneration, contributing to a circular loop linking alterations of proNGF/NGF equilibrium to excitatory/inhibitory synaptic imbalance and amyloid precursor protein (APP) dysmetabolism.Cell Death and Differentiation advance online publication, 29 March 2013; doi:10.1038/cdd.2013.22.
Cell death and differentiation 03/2013; 20(8). DOI:10.1038/cdd.2013.22 · 8.18 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Several open questions call for new studies on pathogenic mechanisms leading to Alzheimer’s Disease (AD), with
the search for upstream drivers of the neurodegeneration cascade, such as neurotrophic deficits, early misfolding
events of AD-related proteins (Aβ and tau) and understanding the multifactorial basis of AD pathogenesis. Since
seminal immunosympathectomy experiment which represents the first example of a knock out experiment (albeit
a protein knock-out), antibodies have had a long and successful history as a tool to selectively interfere with the
function of proteins in cells and in organisms and antibody technologies represent a major weapon in the set
of target validation techniques. Here, we describe a technology, pioneered by our group, based on recombinant
antibody domains exploited as intracellular antibodies (intrabodies) whereby antibodies are used as genes, rather
than as proteins. We discuss several applications and new promising developments of the intrabody approach for
protein interference, especially in the field of AD research.
Journal of biological regulators and homeostatic agents 01/2013; 27(2(S)):89-105. · 2.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nerve Growth Factor (NGF) is being considered as a therapeutic candidate for Alzheimer's disease (AD) treatment but the clinical application is hindered by its potent pro-nociceptive activity. Thus, to reduce systemic exposure that would induce pain, in recent clinical studies NGF was administered through an invasive intracerebral gene-therapy approach. Our group demonstrated the feasibility of a non-invasive intranasal delivery of NGF in a mouse model of neurodegeneration. NGF therapeutic window could be further increased if its nociceptive effects could be avoided altogether. In this study we exploit forms of NGF, mutated at residue R100, inspired by the human genetic disease HSAN V (Hereditary Sensory Autonomic Neuropathy Type V), which would allow increasing the dose of NGF without triggering pain. We show that "painless" hNGF displays full neurotrophic and anti-amyloidogenic activities in neuronal cultures, and a reduced nociceptive activity in vivo. When administered intranasally to APPxPS1 mice ( n = 8), hNGFP61S/R100E prevents the progress of neurodegeneration and of behavioral deficits. These results demonstrate the in vivo neuroprotective and anti-amyloidogenic properties of hNGFR100 mutants and provide a rational basis for the development of "painless" hNGF variants as a new generation of therapeutics for neurodegenerative diseases.
PLoS ONE 05/2012; 7(5):e37555. DOI:10.1371/journal.pone.0037555 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background: Increasing evidence supports the role of intracellular Aß oligomerization as an early event in the pathogenesis of Alzheimer’s Disease in humans and in transgenic mice, but little is known about the intracellular processing and trafficking events of the different forms of AßOs. Here, we propose the use of intracellular antibodies (intrabodies) to dissect the cellular pathways leading to AßOs formation and actions, exploiting new conformational
anti-AßOs antibody fragments (Meli et al., J. Mol. Biol. 2009),
which are suitable for intracellular expression. The use of specific conformational
intrabodies allows interfering in cells with post-translationally
modified, or processed, proteins (as APP and its products Aß/AßOs) in
a way that cannot be performed with the RNAi-based methods. Methods:
In this study, we propose an intrabody-mediated targeting of AßOs in cultured
cells. The intracellular expression of the anti-AßOs intrabodies was
performed in a well established cell model for AßOs production and secretion,
referred to as 7PA2 cells (Walsh et al., Nature, 2002), generating cells
stably transfected with intrabodies in different formats. The in vivo binding
and the modulation of AßOs generation were studied by analysis of cellular
media and by experiments of sub cellular fractionation and intrabody-AßOs
pull-down. Results: The anti-AßOs intrabodies targeted to specific intracellular
compartments act as specific interference tools. In particular, we performed
the specific and selective sub cellular targeting of AßOs in the
Endoplasmic Reticulum (ER), through the expression of an intrabody
with an ER retention signal (KDEL). The use of derivative KDEL form of
an anti-AßOs intrabody shows: i) the efficacy in AßOs-binding, trapping
and retargeting in specific ER compartments; ii) the ability to selectively reduce
the secretion of small AßOs; iii) no interference in the post-translational
maturation of the Aß precursor protein (APP). Conclusions: The
specific sub cellular localization of anti-AßOs intrabodies and their efficacy
in vivo in AßOs-binding, trapping and in the reduction of AßOs secreted
shows that Aß principally do not oligomerize post-secretion, but intracellularly.
Thus, intracellular AßOs are a relevant target for new therapeutics.We
confirmed the anti-AßOs intrabodies as unique tool to study the intracellular
Aß oligomerization and also to be exploited as new diagnostics and
Alzheimer's & dementia: the journal of the Alzheimer's Association 07/2011; 7(4):S391. DOI:10.1016/j.jalz.2011.05.1130 · 12.41 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Synapses are ultrastructural sites for memory storage in brain, and synaptic damage is the best pathologic correlate of cognitive decline in Alzheimer's disease (AD). Post-translational hyperphosphorylation, enzyme-mediated truncation, conformational modifications, and aggregation of tau protein into neurofibrillary tangles (NFTs) are hallmarks for a heterogeneous group of neurodegenerative disorders, so-called tauopathies. AD is a secondary tauopathy since it is pathologically distinguished by the presence of amyloid-beta (Abeta)-containing senile plaques and the presence of tau-positive NFTs in the neocortex and hippocampus. Here, we report that a 20-22 kDa NH2-truncated tau fragment is largely enriched in human mitochondria from cryopreserved synaptosomes of AD brains and that its amount in terminal fields correlates with the pathological synaptic changes and with the organelle functional impairment. This NH2-truncated tau form is also found in other human, not AD-tauopathies, while its presence in AD patients is linked to Abeta multimeric species and likely to pathology severity. Finally native, patient-derived, Abeta oligomers-enriched extracts likely impair the mitochondrial function by the in vitro production of 20-22 kDa NH2-tau fragments in mature human SY5Y and in rat hippocampal neurons. Thus our findings suggest that the mitochondrial NH2-derived tau peptide distribution may exacerbate the synapse degeneration occurring in tauopathies, including AD, and sustain the in vivo NH-2 tau cleavage inhibitors as an alternative drug discovery strategies for AD therapy.
[Show abstract][Hide abstract] ABSTRACT: The development of conformation-sensitive antibody domains targeting the misfolding beta amyloid (Aβ) peptide is of great interest for research into Alzheimer's disease (AD). We describe the direct selection, by the Intracellular Antibody Capture Technology (IACT), of a panel of anti-Aβ single chain Fv antibody fragments (scFvs), targeting pathologically relevant conformations of Aβ. A LexA-Aβ1-42 fusion protein was expressed in yeast cells, as the "intracellular antigen". Two different scFv antibody libraries (Single Pot Libraries of Intracellular Antibodies, SPLINT) were used for the intracellular selections: (i) a naïve library, derived from a natural, non-immune, source of mouse antibody variable region (V) genes; and (ii) an immune library constructed from the repertoire of antibody V genes of Aβ-immunized mice. This led to the isolation of 18 different anti-Aβ scFvs, which bind Aβ both in the yeast cell, as well as in vitro, if used as purified recombinant proteins. Surprisingly, all the anti-Aβ scFvs isolated are conformation-sensitive, showing a high degree of specificity towards Aβ oligomers with respect to monomeric Aβ, while also displaying some degree of sequence-specificity, recognizing either the N-terminal or the C-terminal part of Aβ1-42; in particular, the scFvs selected from Aβ-immune SPLINT library show a relevant N-terminal epitope bias. Representative candidates from this panel of the anti-Aβ scFvs were shown to recognize in vivo-produced Aβ "deposits" in histological sections from human AD brains and to display good neutralization properties, significantly inhibiting Aβ oligomer-induced toxicity and synaptic binding of Aβ oligomers in neuronal cultured cells. The properties of these anti-Aβ antibody domains, as well as their direct availability for intra- or extra-cellular "genetic delivery" make them ideally suited for new experimental approaches to study and image the intracellular processing and trafficking of Aβ oligomers.
[Show abstract][Hide abstract] ABSTRACT: Nerve growth factor (NGF) exerts a trophic, antiapoptotic action on several neuronal targets, including the clonal cell line PC12. In the current study, we demonstrate that withdrawal of this neurotrophin from PC12 differentiated cells causes overproduction of amyloid-beta (Abeta) peptides, which are the most toxic protein fragments directly implicated in the development of Alzheimer disease (AD), concomitantly with cell death by apoptosis. Abeta production and apoptotic death, occurring after withdrawal from NGF-differentiated PC12 cells, are completely inhibited by beta- and gamma-secretase inhibitors and by antibodies directed against Abeta peptides, favouring maintenance of PC12 morphology and neuritic network. These peptides are partially released and largely deposited as aggregates only soluble with strong detergent treatment generally employed to dissolve senile plaques. Furthermore, partial silencing of APP mRNA, by siRNA, reduces not only the extent of Abeta production but also apoptotic death. Abeta production and apoptosis are also induced in differentiated PC12 cells by kinase inhibitors of Trk-A, the high affinity receptor of NGF and, in this case, the co-incubation with beta- and gamma-secretase inhibitors totally revert apoptosis.
[Show abstract][Hide abstract] ABSTRACT: The intracellular antibody technology has many applications for proteomics studies. The potential of intracellular antibodies for the systematic study of the proteome has been made possible by the development of new experimental strategies that allow the selection of antibodies under conditions of intracellular expression. The Intracellular Antibody Capture Technology (IACT) is an in vivo two-hybrid-based method originally developed for the selection of antibodies readily folded for ectopic expression. IACT has been used for the rapid and effective identification of novel antigen-antibody pairs in intracellular compartments and for the in vivo identification of epitopes recognized by selected intracellular antibodies. IACT opens the way to the use of intracellular antibody technology for large-scale applications in proteomics. In its present format, its use is however somewhat limited by the need of a preselection of the input phage antibody libraries on protein antigens or by the construction of an antibody library from mice immunized against the target protein(s), to provide an enriched input library to compensate for the suboptimal efficiency of transformation of the yeast cells. These enrichment steps require expressing the corresponding proteins, which represents a severe bottleneck for the scaling up of the technology. We describe here the construction of a single pot library of intracellular antibodies (SPLINT), a naïve library of scFv fragments expressed directly in the yeast cytoplasm in a format such that antigen-specific intrabodies can be isolated directly from gene sequences, with no manipulation whatsoever of the corresponding proteins. We describe also the isolation from SPLINT of a panel of intrabodies against a number of different proteins. The application of SPLINT on a genome-wide scale should help the systematic study of the functional organization of cell proteome.