Simona Mancini

Nanomedicine for the treatment of Alzheimer's disease

Alzheimers disease affects more than 35 million people worldwide and this number is presumed to double by the year 2050. Currently, there is no efficient therapy for this disorder but a promising approach is represented by nanotechnology, easily multifunctionalizable devices with size in the order of billionth of meter. This review provides a concise survey on the nano-based strategies for Alzheimers disease treatment, aiming at carrying drugs across the blood-brain barrier, in particular to target the metabolism of β-amyloid peptide, a pivotal player in this pathology.

Soluble Aβ oligomer-induced synaptopathy: c-Jun N-terminal kinase's role

Dear Editor, Among the neurodegenerative diseases, Alzheimer disease (AD) is the most common and severe age-related dementia for which there is currently no available treatment. Many studies support the assumption that AD is a spine pathology (Selkoe, 2002; Sivanesan et al., 2013) and that soluble amyloid-b (Ab) oligomers are causative of AD synaptopathy. Diverse lines of evidence indicate that Aboligomers induce formation of pore-like structures on the membrane (Arispe et al., 1993; Lashuel et al., 2002) and interfere with glutamatergic transmission. The Ab oligomers result in a decreased number of AMPA receptors (AMPA-r) and NMDA receptors (NMDA-r), as well as PSD-95 at the postsynaptic membrane, and thus reduce the strength and plasticity of excitatory synapses (Chapman et al., 1999; Walsh et al., 2002). However, the underlying intracellular mechanisms regulating synaptic changes are only partially known. By understanding the pathophysiological mechanisms leading to synaptic dysfunction and the progression of this dysfunction, better interference in the pathogenesis of AD can be achieved. We present an in vitro model to study the temporal sequence of dendritic spine modifications induced by soluble Ab oligomers, and to analyse the intracellular signalling pathways leading to AD synaptopathy. This model allows synaptic alterations to be followed in living neurons before and after treatment and reduces bias due to cell variability. This model also permits testing of pharmaceuticals that are designed to reverse the biochemical and structural alterations of synapses induced by Ab oligomers. Brainbow hippocampal neurons, which express fluorescent proteins, were used to visualize dendritic spines and study synaptic plasticity (Figure 1A and B). To obtain isolated cells, fluorescent neurons were seeded on a layer of non-fluorescent neurons (ratio: 1/16) (Figure 1A and B). In this way we avoided the need for transfection, infection protocols, and low density cultures that are not well tolerated by neurons. Neurons were treated with a subtoxic dose (Figure 1E) of soluble Ab1–42 oligomers in order to induce synaptic changes without any signs of neuronal death (Figure 1E). The preparation of synthetic Ab1–42 that were used to induce in vitro synaptic dysfunction had been previously characterized. Oligomeric assemblies were only observed in peptide preparations after a 24-h incubation at 48C (referred to as oligomers). Immediately after dissolution, the majority of Ab1–42 remained as unassembled monomeric structures (Figure 1C and D and Supplementary Figure S1). Subtoxic concentrations of soluble Ab oligomers induced alterations in the postsynaptic density (PSD) composition of dendritic spines, while monomers had no effect on synaptic plasticity (Supplementary Figure S1F). Exposing the neurons for 3 h to 1 mM Ab oligomers induced changes in the PSD region, leading to a 68% and 61% drop of GluN2A and GluN2B subunits of NMDA-r, respectively; a decrease of 70% and 65% of GluA1 and GluA2 subunits of AMPA-r, respectively; a 53% loss of PSD-95; and a 76% loss of drebrin (Figure 1F and Supplementary Figure S2A). To assess dendritic spine modifications in vitro, we analysed changes in spine density and morphology in neurons exposed to soluble Ab oligomers. Application of soluble Ab oligomers (1 mM) for 3 h caused a 25% decrease in total spine number compared with that before Ab application (Figure 1J and K). The decrease involved all types of spines in a proportional manner. The number of mushroom, stubby, and thin spines decreased by 32%, 25%, and 22%, respectively (Figure 1J and L). Moreover, Ab oligomer treatment induced a 67% reduction in new spine formation compared with control conditions, and led to spine shrinkage (Supplementary Figure S2E). The number of mushroom spines that became stubby or thin was significantly increased by Ab oligomer treatment, while the number of spines that became mushroom was decreased (Supplementary Figure S2E). The morphological changes were consistent with the observed biochemical alterations since thin and stubby spines have a less extended PSD region and lower contents of glutamate receptors as well as postsynaptic markers, in comparison with mushroom spines (Tackenberg et al., 2009). To analyse the pathways involved in Ab oligomer-induced synaptopathy, we evaluated synaptic changes induced after 30 min and 3 h of Ab oligomer exposure (1 mM) and correlated them to the activation of two stress signalling pathways, c-Jun N-terminal kinase (JNK) and caspase-3. After 30 min there was no sign of molecular changes: NMDA-r and AMPA-r subunits, PSD-95, and drebrin levels were unaffected by the Ab oligomer treatment (Figure 1F). However, JNK was already activated at this stage, as indicated by a 2.36-fold increase of the P-JNK/JNK ratio compared with control conditions (Figure 1G and Supplementary Figure S2B). There was no indication of caspase-3 cleavage after 30 min of Ab oligomer exposure (Figure 1H and Supplementary Figure S2C). Exposing neurons to soluble Ab oligomers for 3 h induced a biochemical perturbation of PSD (Figure 1F) with a 4.33-fold increase of caspase-3 cleavage (Figure 1H) (Li et al., 2010; D’Amelio et al., 2011), while JNK activity remained elevated by 2.26 folds (Figure 1G). Our results showed that JNK activation was triggered by Ab oligomers before PSD alterations were induced and JNK activation persisted up to 3 h, at which doi:10.1093/jmcb/mjt015 Journal of Molecular Cell Biology (2013), 5, 277–279 | 277 Published online April 18, 2013

Retro-inverso peptide inhibitor nanoparticles as potent inhibitors of aggregation of the Alzheimer’s Aβ peptide

Aggregation of amyloid-β peptide (Aβ) is a key event in the pathogenesis of Alzheimers disease (AD). We investigated the effects of nanoliposomes decorated with the retro-inverso peptide RI-OR2-TAT (Ac-rGffvlkGrrrrqrrkkrGy-NH2) on the aggregation and toxicity of Aβ. Remarkably low concentrations of these peptide inhibitor nanoparticles (PINPs) were required to inhibit the formation of Aβ oligomers and fibrils in vitro, with 50% inhibition occurring at a molar ratio of ~1:2000 of liposome-bound RI-OR2-TAT to Aβ. PINPs also bound to Aβ with high affinity (Kd=13.2-50 nM), rescued SHSY-5Y cells from the toxic effect of pre-aggregated Aβ, crossed an in vitro blood-brain barrier model (hCMEC/D3 cell monolayer), entered the brains of C57 BL/6 mice, and protected against memory loss in APPSWE transgenic mice in a novel object recognition test. As the most potent aggregation inhibitor that we have tested so far, we propose to develop PINPs as a potential disease-modifying treatment for AD.

Multifunctional liposomes delay phenotype progression and prevent memory impairment in a presymptomatic stage mouse model of Alzheimer disease

ABSTRACT The failure of clinical trials largely focused on mild to moderate stages of Alzheimer disease has suggested to the scientific community that the effectiveness of Amyloid‐&bgr; (A&bgr;)‐centered treatments should be evaluated starting as early as possible, well before irreversible brain damage has occurred. Accordingly, also the preclinical development of new therapies should be carried out taking into account this suggestion. In the present investigation we evaluated the efficacy of a treatment with liposomes multifunctionalized for crossing the blood‐brain barrier and targeting A&bgr;, carried out on young APP/PS1 Tg mice, taken as a model of pre‐symptomatic disease stage. Liposomes were administered once a week to Tg mice for 7 months, starting at the age of 5 months and up to the age of 12 when they display AD‐like cognitive and brain biochemical/anatomical features. The treatment prevented the onset of the long‐term memory impairment and slowed down the deposition of brain A&bgr;; at anatomical level, prevented both ventricle enlargement and entorhinal cortex thickness reduction, otherwise occurring in untreated mice. Strikingly, these effects were maintained 3 months after treatment discontinuation. An increase of A&bgr; levels in the liver was detected at the end of the treatment, then followed also by reduction of brain Amyloid Precursor Protein and increase of A&bgr;‐degrading enzymes. These results suggest that the treatment promotes brain A&bgr; clearance by a peripheral ‘sink’ effect and ultimately affects A&bgr; turnover in the brain. Worth of note, the treatment was apparently not toxic for all the organs analyzed, in particular for brain, as suggested by the lower brain TNF‐&agr; and MDA levels, and by higher level of SOD activity in treated mice. Together, these findings promote a very early treatment with multi‐functional liposomes as a well‐tolerated nanomedicine‐based approach, potentially suitable for a disease‐modifying therapy of AD, able to delay or prevent relevant features of the disease.

The cell-permeable Aβ1-6A2VTAT(D) peptide reverts synaptopathy induced by Aβ1-42wt

Alzheimer disease (AD) is the most prevalent form of dementia. Loss of hippocampal synapses is the first neurodegenerative event in AD. Synaptic loss has been associated with the accumulation in the brain parenchyma of soluble oligomeric forms of amyloid β peptide (Aβ1-42wt). Clinical observations have shown that a mutation in the APP protein (A673V) causes an early onset AD-type dementia in homozygous carriers while heterozygous carriers are unaffected. This mutation leads to the formation of mutated Aβ peptides (Aβ1-42A2V) in homozygous patients, while in heterozygous subjects both Aβ1-42wt and Aβ1-42A2V are present. To better understand the impact of the A673V mutation in AD, we analyzed the synaptotoxic effect of oligomers formed by aggregation of different Aβ peptides (Aβ1-42wt or Aβ1-42A2V) and the combination of the two Aβ1-42MIX (Aβ1-42wt and Aβ1-42A2V) in an in vitro model of synaptic injury. We showed that Aβ1-42A2V oligomers are more toxic than Aβ1-42wt oligomers in hippocampal neurons, confirming the results previously obtained in cell lines. Furthermore, we reported that oligomers obtained by the combination of both wild type and mutated peptides (Aβ1-42MIX) did not exert synaptic toxicity. We concluded that the combination of Aβ1-42wt and Aβ1-42A2V peptides hinders the toxicity of Aβ1-42A2V and counteracts the manifestation of synaptopathy in vitro. Finally we took advantage of this finding to generate a cell-permeable peptide for clinical application, by fusing the first six residues of the Aβ1-42A2V to the TAT cargo sequence (Aβ1-6A2VTAT(D)). Noteworthy, the treatment with Aβ1-6A2VTAT(D) confers neuroprotection against both in vitro and in vivo synaptopathy models. Therefore Aβ1-6A2VTAT(D) may represent an innovative therapeutic tool to prevent synaptic degeneration in AD.

Functionalized liposomes and phytosomes loading Annona muricata L. aqueous extract: Potential nanoshuttles for brain-delivery of phenolic compounds

BACKGROUND Multi-target drugs have gained significant recognition for the treatment of multifactorial diseases such as depression. Under a screening study of multi-potent medicinal plants with claimed antidepressant-like activity, the phenolic-rich Annona muricata aqueous extract (AE) emerged as a moderate monoamine oxidase A (hMAO-A) inhibitor and a strong hydrogen peroxide (H2O2) scavenger. PURPOSE In order to protect this extract from gastrointestinal biotransformation and to improve its permeability across the blood-brain barrier (BBB), four phospholipid nanoformulations of liposomes and phytosomes functionalized with a peptide ligand promoting BBB crossing were produced. METHODS AE and nanoformulations were characterized by HPLC-DAD-ESI-MSn, HPLC-DAD, spectrophotometric, fluorescence and dynamic light scattering methods. Cytotoxicity and permeability studies were carried out using an in vitro transwell model of the BBB, composed of immortalized human microvascular endothelial cells (hCMEC/D3), and in vitro hMAO-A inhibition and H2O2 scavenging activities were performed with all samples. RESULTS The encapsulation/binding of AE was more efficient with phytosomes, while liposomes were more stable, displaying a slower extract release over time. In general, phytosomes were less toxic than liposomes in hCMEC/D3 cells and, when present, cholesterol improved the permeability across the cell monolayer of all tested nanoformulations. All nanoformulations conserved the antioxidant potential of AE, while phosphatidylcholine interfered with MAO-A inhibition assay. CONCLUSIONS Overall, phytosome formulations registered the best performance in terms of binding efficiency, enzyme inhibition and scavenging activity, thus representing a promising multipotent phenolic-rich nanoshuttle for future in vivo depression treatment.

The ability of liposomes, tailored for blood-brain barrier targeting, to reach the brain is dramatically affected by the disease state

AIM To investigate if and how the ability of liposomes, previously designed for Alzheimers therapy, to reach the brain changes in aging/pathological conditions with respect to the healthy state. METHODS Biodistribution and pharmacokinetics of liposomes in young or aged healthy mice and in an Alzheimers mouse model were measured by radiochemical techniques. The expression of brain receptors and structural proteins was evaluated by Western blot. RESULTS At equal blood levels, the amount and integrity of liposomes in the brain were dramatically lower in Alzheimers or aged mice, with respect to young animals. These differences are likely attributable to molecular alterations in the brain vasculature. CONCLUSION Brain alterations in pathology or aging should be considered in the design of drug delivery systems for brain targeting.

Targeting ß-amyloid by the A2V Aß variant: a novel disease-modifying strategy for the treatment of Alzheimer’s disease

Abstracts of the IX Congresso SindemItalian Association for the study of Dementia linked to the Italian Neurological Society (SIN)Firenze, Palazzo dei Congressi, Villa Vittoria March 13-15, 2014Comitato Scientifico: Vincenzo Bonavita, Alessandro Padovani, Amalia Bruni, Leonardo Pantoni, Carlo Caltagirone, Lucilla Parnetti, Francesca Clerici, Daniela Perani, Monica Di Luca, Sandro Sorbi, Gianluigi Forloni, Francesco Tagliavini, Giovanni Frisoni, Marilu Gorno Tempini, Claudio Mariani, Annalena Venneri, Massimo Musicco.