Gabriele Ugolini

The Neuronal Microtubule-Associated Protein Tau Is a Substrate for Caspase-3 and an Effector of Apoptosis

We have identified a class of tau fragments inducing apoptosis in different cellular contexts, including a human teratocarcinoma‐derived cell line (NT2 cells) representing committed human neuronal precursors. We have found a transition point inside the tau molecule beyond which the fragments lose their ability to induce apoptosis. This transition point is located around one of the putative caspase‐3 cleavage sites. This is the only site that can be effectively used by caspase‐3 in vitro, releasing the C‐terminal 19 amino acids of tau. These results establish tau as a substrate for an apoptotic protease that turns tau itself into an effector of apoptosis. Accordingly, tau may be involved in a self‐propagating process like what has been predicted for the pathogenesis of different neurodegenerative disorders.

The function neutralizing anti-TrkA antibody MNAC13 reduces inflammatory and neuropathic pain.

Nerve growth factor (NGF) is involved in pain transduction mechanisms and plays a key role in many persistent pain states, notably those associated with inflammation. On this basis, both the NGF ligand and its receptor TrkA (tyrosine kinase A) represent an eligible target for pain therapy. Although the direct involvement of NGF in pain modulation is well established, the effect of a direct functional block of the TrkA receptor is still unknown. In this study, we have demonstrated that MNAC13, the only anti-TrkA monoclonal antibody for which function neutralizing properties have been clearly shown both in vitro and in vivo, induces analgesia in both inflammatory and neuropathic pain models, with a surprisingly long-lasting effect in the latter. The formalin-evoked pain licking responses are significantly reduced by the MNAC13 antibody in CD1 mice. Remarkably, treatment with the anti-TrkA antibody also produces a significant antiallodynic effect on neuropathic pain: repeated i.p. injections of MNAC13 induce significant functional recovery in mice subjected to sciatic nerve ligation, with effects persisting after administration. Furthermore, a clear synergistic effect is observed when MNAC13 is administered in combination with opioids, at doses that are not efficacious per se. This study represents a direct demonstration that neutralizing antibodies directed against the TrkA receptor may display potent analgesic effects in inflammatory and chronic pain.

Apoptotic effect of caspase-3 cleaved tau in hippocampal neurons and its potentiation by tau FTDP-mutation N279K

Pathological changes in the microtubule associated protein tau are a major hallmark of many human dementias collectively defined as tauopathies. In familiar frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), several mutations in the tau gene have been identified showing that primary malfunction of tau can lead to neurodegeneration. In addition to mutation at genetic level, a number of post-translational modifications of tau occur in tauopathies, including abnormal phosphorylation and aberrant proteolysis described in Alzheimers Disease (AD). The presence of cleaved tau in AD neurons is associated with expression of markers for neuronal death. According to our previous work, tau is a substrate for the apoptotic protease caspase-3 that turns tau itself into an effector of apoptosis (tau cleaved at D-421), generating a positive-feedback loop that is self-propagating. Cleavage of tau by caspase-3 was recently confirmed to occur in AD brain as an early event. Here we show the apoptotic properties of tau fragment tau151-421 in primary cultures of rat hippocampal neurons; such cellular model is of special interest considering the selective vulnerability of hippocampal neurones in AD. The apoptotic capacity of tau151-421 is markedly enhanced by both treatment with amyloid peptide Abeta25-35, and the FTDP-17 tau mutation N279K.

Taking Pain Out of NGF: A “Painless” NGF Mutant, Linked to Hereditary Sensory Autonomic Neuropathy Type V, with Full Neurotrophic Activity

During adulthood, the neurotrophin Nerve Growth Factor (NGF) sensitizes nociceptors, thereby increasing the response to noxious stimuli. The relationship between NGF and pain is supported by genetic evidence: mutations in the NGF TrkA receptor in patients affected by an hereditary rare disease (Hereditary Sensory and Autonomic Neuropathy type IV, HSAN IV) determine a congenital form of severe pain insensitivity, with mental retardation, while a mutation in NGFB gene, leading to the aminoacid substitution R100W in mature NGF, determines a similar loss of pain perception, without overt cognitive neurological defects (HSAN V). The R100W mutation provokes a reduced processing of proNGF to mature NGF in cultured cells and a higher percentage of neurotrophin secreted is in the proNGF form. Moreover, using Surface Plasmon Resonance we showed that the R100W mutation does not affect NGF binding to TrkA, while it abolishes NGF binding to p75NTR receptors. However, it remains to be clarified whether the major impact of the mutation is on the biological function of proNGF or of mature NGF and to what extent the effects of the R100W mutation on the HSAN V clinical phenotype are developmental, or whether they reflect an impaired effectiveness of NGF to regulate and mediate nociceptive transmission in adult sensory neurons. Here we show that the R100 mutation selectively alters some of the signaling pathways activated downstream of TrkA NGF receptors. NGFR100 mutants maintain identical neurotrophic and neuroprotective properties in a variety of cell assays, while displaying a significantly reduced pain-inducing activity in vivo (n = 8–10 mice/group). We also show that proNGF has a significantly reduced nociceptive activity, with respect to NGF. Both sets of results jointly contribute to elucidating the mechanisms underlying the clinical HSAN V manifestations, and to clarifying which receptors and intracellular signaling cascades participate in the pain sensitizing action of NGF.

Delivery of NGF to the brain: intranasal versus ocular administration in anti-NGF transgenic mice.

Nerve growth factor (NGF) has a great potential for the treatment of Alzheimers disease. However, the therapeutic administration of NGF represents a significant challenge, due to the difficulty to deliver relevant doses to the brain, in a safe and non-invasive way. We previously demonstrated the efficacy of a non-invasive delivery of NGF to the brain in animal models, by an intranasal route. Recently, topical eye application of NGF was proposed, as an option for the delivery of NGF to the brain. Here, we compare the efficacy of the two delivery routes of hNGF-61, a recombinant traceable form of human NGF, in the mouse neurodegeneration model AD11. The intranasal administration appeared to be significantly more effective than the ocular one, in rescuing the neurodegenerative phenotypic hallmarks in AD11 mice. The ocular administration of hNGF-61 showed a more limited efficacy, even at higher doses. Thus, NGF nasal drops represent a viable and effective option to successfully deliver therapeutic NGF to the brain in a non-invasive manner.

Co-localization of truncated tau and Dna fragmentation in Alzheimer's disease neurones

MONOCLONAL antibody MN423 stains all hallmarks of tau neurofibrillary pathology in Alzheimers disease (AD), without reacting with full-length tau. It recognizes tau molecules which are cleaved at Glu-391, suggesting that tau is endogenously truncated in AD brains. We investigated whether truncation of tau takes place in pre-tangle neurones. Our results showed that tau truncation occurs inside AD neurones in the absence of other signs of AD neurofibrillary pathology. Using in situ end labelling (ISEL) of DNA we found evidence for DNA fragmentation in a significant subpopulation of MN423-positive neurones. Our results therefore suggest that tau truncation is an early intracellular event preceding cell death in AD.

Single cycle structure-based humanization of an anti-nerve growth factor therapeutic antibody.

Most forms of chronic pain are inadequately treated by present therapeutic options. Compelling evidence has accumulated, demonstrating that Nerve Growth Factor (NGF) is a key modulator of inflammatory and nociceptive responses, and is a promising target for the treatment of human pathologies linked to chronic and inflammatory pain. There is therefore a growing interest in the development of therapeutic molecules antagonising the NGF pathway and its nociceptor sensitization actions, among which function-blocking anti-NGF antibodies are particularly relevant candidates. In this respect, the rat anti-NGF αD11 monoclonal antibody (mAb) is a potent antagonist, able to effectively antagonize rodent and human NGF in a variety of in vitro and in vivo systems. Here we show that mAb αD11 displays a significant analgesic effect in two different models of persistent pain in mice, with a remarkable long-lasting activity. In order to advance αD11 mAb towards its clinical application in man, anti-NGF αD11 mAb was humanized by applying a novel single cycle strategy based on the a priori experimental determination of the crystal and molecular structure of the parental Fragment antigen-binding (Fab). The humanized antibody (hum-αD11) was tested in vitro and in vivo, showing that the binding mode and the NGF neutralizing biological activities of the parental antibody are fully preserved, with even a significant affinity improvement. The results firmly establish hum-αD11 as a lead candidate for clinical applications in a therapeutic area with a severe unmet medical need. More generally, the single-cycle structure-based humanization method represents a considerable improvement over the standard humanization methods, which are intrinsically empirical and require several refinement cycles.

P2-424: Delivery of NGF to the brain: Intranasal versus ocular administration in transgenic mice

Background: The concept of therapeutic administration of human recombinant Nerve Growth Factor (NGF) in AD patients has been well validated by preclinical and clinical studies. However, a significant challenge to the clinical use of NGF is the difficulty associated with its delivery to the brain at therapeutically relevant doses. Indeed, NGF is not orally available and unable to access the blood-brain barrier, following parenteral systemic administration. Clinical studies performed so far were based on intracerebroventricular administration or on a gene therapy based on grafting in the brain parenchyma of cells engineered to secrete NGF. Although encouraging in terms of efficacy, these results can be questioned in terms of widespread clinical applicability, due to the invasiveness of the surgical procedure. A non invasive approach for NGF delivery to the brain would be highly desirable. Our laboratory already obtained a proof of concept for the feasibility of a non invasive strategy to delivery NGF to the brain, by treating intranasally a model of Alzheimer’s disease (the AD11 anti-NGF mouse) and obtaining in such a way the rescue of behavioral deficits and neurodegeneration (Capsoni et al 2002; De Rosa et al, 2005). Recently, it was shown that topical eye application of NGF causes an enhanced expression ChAT immunoreactivity in rat basal forebrain cholinergic neurons, suggesting that ocular NGF application might be a viable option for the effective delivery of NGF to the brain (Lambiase et al., 2006). Here, we compare the efficacy of intranasal versus ocular administration of NGF in transgenic AD11 mice. Methods: By using a new analog of human NGF (hNGF-61), characterized by a single amino acid mutation that allows it to be detected against hNGF background, we compared the efficacy of the two methods of administration in rescuing behavioral deficits and neurodegeneration in AD11 mice. Results: Although the ocular administration of hNGF-61 showed a certain extent of efficacy, the intranasal administration appeared to be significantly more effective in rescuing the whole set of phenotypic hallmarks in AD11 mice, even at lower doses. Conclusions: Thus, NGF nasal drops can represent a non-invasive strategy to successfully deliver NGF to the brain.

P2-380: Novel recombinant antibodies against AD biomarkers linked to neurotrophin signalling as potential diagnostic tools

achieve a uniform quality level for this study, the dataset was reduced to 224 subjects from 6 centers. A supervised fully connected ANN was implemented with a single layer of hidden units and 30 inputs (3 modalities, each with their respective 3x3 neighborhood and spatial information). The output comprised of four probability maps GM, WM, CSF and ARWMC, but only the ARWMC output is assessed here. Before segmentation all data were pre-processed for head motion and intensity variations. The ANN was trained with an ROI dataset drawn on 6 subjects from 4 centers. For segmentation, the optimal threshold of the ARWMC probability map was determined using the Index of Similarity describing the overlap between ANN and manual ROI’s. Here a threshold of 90% was found to be optimal. Results: From each of the 6 center the correlation coefficient between ANN and manually determined were generally high: 0.97, 0.97, 0.87, 0.88, 0.73 and 0.98. The correlation of the combined set was 0.89. The two first centers, with a correlation coefficient of 0.97, had two subjects in the training set which may explain their better performance. Deviating quality in the FLAIR image contributes to the lowest correlation in a center. In other cases a systematic bias was present in some anatomical regions (e.g. septum pellucidum, where lesion were not drawn manually) or regarding lacunar infarcts. Conclusion: In this multi-center investigation of ARWMC segmentation a high average correlation of 0.89 was found. Improvements may be gained from representing more centres in the training set. The results demonstrate the generalizability of ANN methods, and underline the importance of rigorous standardization of MRI quality in multi-center studies.

The neuronal microtubule associated protein tau is a substrate for caspase-3 and an effector of apoptosis

We have identified a class of tau fragments inducing apoptosis in different cellular contexts, including a human teratocarcinoma-derived cell line (NT2 cells) representing committed human neuronal precursors. We have found a transition point inside the tau molecule beyond which the fragments lose their ability to induce apoptosis. This transition point is located around one of the putative caspase-3 cleavage sites. This is the only site that can be effectively used by caspase-3 in vitro, releasing the C-terminal 19 amino acids of tau. These results establish tau as a substrate for an apoptotic protease that turns tau itself into an effector of apoptosis. Accordingly, tau may be involved in a self-propagating process like what has been predicted for the pathogenesis of different neurodegenerative disorders.