Giovanni Meli

Activation of the Amyloidogenic Route by NGF Deprivation Induces Apoptotic Death in PC12 Cells

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.

A NH2 tau fragment targets neuronal mitochondria at AD synapses: possible implications for neurodegeneration.

Synapses are ultrastructural sites for memory storage in brain, and synaptic damage is the best pathologic correlate of cognitive decline in Alzheimers 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.

Intracellular antibodies for proteomics

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 naı̈ve 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.

Intranasal “painless” Human Nerve Growth Factors Slows Amyloid Neurodegeneration and Prevents Memory Deficits in App X PS1 Mice

Nerve Growth Factor (NGF) is being considered as a therapeutic candidate for Alzheimers 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.

ProNGF\NGF imbalance triggers learning and memory deficits, neurodegeneration and spontaneous epileptic-like discharges in transgenic mice

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.

Conformational targeting of intracellular Aβ oligomers demonstrates their pathological oligomerization inside the endoplasmic reticulum

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.

Characterization of Mitochondrial Dysfunction in the 7PA2 Cell Model of Alzheimer's Disease

The 7WD4 and 7PA2 cell lines, widely used as cellular models for Alzheimers 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.

The chemokine CXCL12 mediates the anti-amyloidogenic action of painless human nerve growth factor

Nerve growth factor is a therapeutic candidate for Alzheimer’s disease. Due to its pain-inducing activity, in current clinical trials nerve growth factor is delivered locally into the brain by neurosurgery, but data on the efficacy of local nerve growth factor delivery in decreasing amyloid-&bgr; deposition are not available. To reduce the nerve growth factor pain-inducing side effects, thus avoiding the need for local brain injection, we developed human painless nerve growth factor (hNGFp), inspired by the human genetic disease hereditary sensory and autonomic neuropathy type V. hNGFp has identical neurotrophic potency as wild-type human nerve growth factor, but a 10-fold lower pain sensitizing activity. In this study we first mimicked, in the 5xFAD mouse model, the intraparenchymal delivery of hNGFp used in clinical trials and found it to be ineffective in decreasing amyloid-&bgr; plaque load. On the contrary, the same dose of hNGFp delivered intranasally, which was widely biodistributed in the brain and did not induce pain, showed a potent anti-amyloidogenic action and rescued synaptic plasticity and memory deficits. We found that hNGFp acts on glial cells, modulating inflammatory proteins such as the soluble TNF&agr; receptor II and the chemokine CXCL12. We further established that the rescuing effect by hNGFp is mediated by CXCL12, as pharmacological inhibition of CXCL12 receptor CXCR4 occludes most of hNGFp effects. These findings have significant therapeutic implications: (i) we established that a widespread exposure of the brain is required for nerve growth factor to fully exert its neuroprotective actions; and (ii) we have identified a new anti-neurodegenerative pathway as a broad target for new therapeutic opportunities for neurodegenerative diseases.

NGF steers microglia toward a neuroprotective phenotype

Microglia are the sentinels of the brain but a clear understanding of the factors that modulate their activation in physiological and pathological conditions is still lacking. Here we demonstrate that Nerve Growth Factor (NGF) acts on microglia by steering them toward a neuroprotective and anti‐inflammatory phenotype. We show that microglial cells express functional NGF receptors in vitro and ex vivo. Our transcriptomic analysis reveals how, in primary microglia, NGF treatment leads to a modulation of motility, phagocytosis and degradation pathways. At the functional level, NGF induces an increase in membrane dynamics and macropinocytosis and, in vivo, it activates an outward rectifying current that appears to modulate glutamatergic neurotransmission in nearby neurons. Since microglia are supposed to be a major player in Aβ peptide clearance in the brain, we tested the effects of NGF on its phagocytosis. NGF was shown to promote TrkA‐mediated engulfment of Aβ by microglia, and to enhance its degradation. Additionally, the proinflammatory activation induced by Aβ treatment is counteracted by the concomitant administration of NGF. Moreover, by acting specifically on microglia, NGF protects neurons from the Aβ‐induced loss of dendritic spines and inhibition of long term potentiation. Finally, in an ex‐vivo setup of acute brain slices, we observed a similar increase in Aβ engulfment by microglial cells under the influence of NGF. Our work substantiates a role for NGF in the regulation of microglial homeostatic activities and points toward this neurotrophin as a neuroprotective agent in Aβ accumulation pathologies, via its anti‐inflammatory activity on microglia.

IDENTIFICATION OF INFLAMMATORY PROCESSES SURROUNDING AMYLOID-β DEPOSITS IN THE RETINA OF MCI AND AD PATIENTS

P4-254 IDENTIFICATION OF INFLAMMATORY PROCESSES SURROUNDING AMYLOID-b DEPOSITS INTHERETINAOFMCIANDAD PATIENTS Koronyo-Hamaoui Maya, Altan Rentsendorj, Ernesto Barron, DieuTrang Fuchs, Carol A. Miller, Keith L. Black, David R. Hinton, Giovanni Meli, Yosef Koronyo, Department of Neurosurgery, Maxine Dunitz Neurosurgical Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA; Department of Biomedical Sciences, Maxine Dunitz Neurosurgical Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA; Doheney Eye Institute, Los Angeles, CA, USA; Department of Pathology Program in Neuroscience, Keck School of Medicine, USC, Los Angeles, CA, USA; Departments of Pathology and Ophthalmology, USC Roski Eye Institute, Keck School of Medicine, USC, Los Angeles, CA, USA; European Brain Research Institute (EBRI), Rome, Italy. Contact e-mail: Maya.Koronyo@cshs.org