A blood-brain barrier (BBB) disrupter is also a potent α-synuclein (α-syn) aggregation inhibitor: a novel dual mechanism of mannitol for the treatment of Parkinson disease (PD).

The development of disease-modifying therapy for Parkinson disease has been a main drug development challenge, including the need to deliver the therapeutic agents to the brain. Here, we examined the ability of mannitol to interfere with the aggregation process of α-synuclein in vitro and in vivo in addition to its blood-brain barrier-disrupting properties. Using in vitro studies, we demonstrated the effect of mannitol on α-synuclein aggregation. Although low concentration of mannitol inhibited the formation of fibrils, high concentration significantly decreased the formation of tetramers and high molecular weight oligomers and shifted the secondary structure of α-synuclein from α-helical to a different structure, suggesting alternative potential pathways for aggregation. When administered to a Parkinson Drosophila model, mannitol dramatically corrected its behavioral defects and reduced the amount of α-synuclein aggregates in the brains of treated flies. In the mThy1-human α-synuclein transgenic mouse model, a decrease in α-synuclein accumulation was detected in several brain regions following treatment, suggesting that mannitol promotes α-synuclein clearance in the cell bodies. It appears that mannitol has a general neuroprotective effect in the transgenic treated mice, which includes the dopaminergic system. We therefore suggest mannitol as a basis for a dual mechanism therapeutic agent for the treatment of Parkinson disease.

Spatial regulation of cell adhesion in the Drosophila wing is mediated by Delilah, a potent activator of ßPS integrin expression.

In spite of our conceptual view of how differential gene expression is used to define different cell identities, we still do not understand how different cell identities are translated into actual cell properties. The example discussed here is that of the fly wing, which is composed of two main cell types: vein and intervein cells. These two cell types differ in many features, including their adhesive properties. One of the major differences is that intervein cells express integrins, which are required for the attachment of the two wing layers to each other, whereas vein cells are devoid of integrin expression. The major signaling pathways that divide the wing to vein and intervein domains have been characterized. However, the genetic programs that execute these two alternative differentiation programs are still very roughly drawn. Here we identify the bHLH protein Delilah (Dei) as a mediator between signaling pathways that specify intervein cell-fate and one of the most significant realizators of this fate, ßPS integrin. Dei's expression is restricted to intervein territories where it acts as a potent activator of ßPS integrin expression. In the absence of normal Dei activity the level of ßPS integrin is reduced, leading to a failure of adhesion between the dorsal and ventral wing layers and a consequent formation of wing blisters. The effect of Dei on ßPS expression is not restricted to the wing, suggesting that Dei functions as a general genetic switch, which is turned on wherever a sticky cell-identity is determined and integrin-based adhesion is required.

The proprioceptive and contractile systems in Drosophila are both patterned by the EGR family transcription factor Stripe.

Coordinated locomotion of Drosophila larvae depends on accurate patterning and stable attachment to the cuticle of both muscles and proprioceptors (chordotonal organs). Unlike muscle spindles in mammals, the fly chordotonal organs are not embedded in the body-wall muscles. Yet, the contractile system (muscles and tendons) and the chordotonal organs constitute two parts of a single functional unit that controls locomotion, and thus must be patterned in full coordination. It is not known how such coordination is achieved. Here we show that the positioning and differentiation of the migrating chordotonal organs are instructed by Stripe, the same transcription factor that promotes tendon cell specification and differentiation and is required for normal patterning of the contractile system. Our data demonstrate that although chordotonal organs are patterned in a Stripe-dependent mechanism similarly to muscles, this mechanism is independent of Stripe activity in tendon cells. Thus, the two parts of the locomotive system use similar but independent patterning mechanisms that converge to form a functional unit. Stripe plays at least a dual role in chordotonal development. It is required within the ligament cells for terminal differentiation and proper migration, without which no induction of ligament attachment cells takes place. Stripe's activity is then necessary within the recruited cells for their differentiation as attachment cells. Similarly to the biphasic differentiation program of tendons, terminal differentiation of chordotonal attachment cells is associated with sequential activation of the two Stripe isoforms-Stripe B and Stripe A.

Inhibiting α-synuclein oligomerization by stable cell-penetrating ß-synuclein fragments recovers phenotype of Parkinson's disease model flies.

The intracellular oligomerization of α-synuclein is associated with Parkinson's disease and appears to be an important target for disease-modifying treatment. Yet, to date, there is no specific inhibitor for this aggregation process. Using unbiased systematic peptide array analysis, we identified molecular interaction domains within the ß-synuclein polypeptide that specifically binds α-synuclein. Adding such peptide fragments to α-synuclein significantly reduced both amyloid fibrils and soluble oligomer formation in vitro. A retro-inverso analogue of the best peptide inhibitor was designed to develop the identified molecular recognition module into a drug candidate. While this peptide shows indistinguishable activity as compared to the native peptide, it is stable in mouse serum and penetrates α-synuclein over-expressing cells. The interaction interface between the D-amino acid peptide and α-synuclein was mapped by Nuclear Magnetic Resonance spectroscopy. Finally, administering the retro-inverso peptide to a Drosophila model expressing mutant A53T α-synuclein in the nervous system, resulted in a significant recovery of the behavioral abnormalities of the treated flies and in a significant reduction in α-synuclein accumulation in the brains of the flies. The engineered retro-inverso peptide can serve as a lead for developing a novel class of therapeutic agents to treat Parkinson's disease.

Complete phenotypic recovery of an Alzheimer's disease model by a quinone-tryptophan hybrid aggregation inhibitor.

The rational design of amyloid oligomer inhibitors is yet an unmet drug development need. Previous studies have identified the role of tryptophan in amyloid recognition, association and inhibition. Furthermore, tryptophan was ranked as the residue with highest amyloidogenic propensity. Other studies have demonstrated that quinones, specifically anthraquinones, can serve as aggregation inhibitors probably due to the dipole interaction of the quinonic ring with aromatic recognition sites within the amyloidogenic proteins. Here, using in vitro, in vivo and in silico tools we describe the synthesis and functional characterization of a rationally designed inhibitor of the Alzheimer's disease-associated beta-amyloid. This compound, 1,4-naphthoquinon-2-yl-L-tryptophan (NQTrp), combines the recognition capacities of both quinone and tryptophan moieties and completely inhibited Abeta oligomerization and fibrillization, as well as the cytotoxic effect of Abeta oligomers towards cultured neuronal cell line. Furthermore, when fed to transgenic Alzheimer's disease Drosophila model it prolonged their life span and completely abolished their defective locomotion. Analysis of the brains of these flies showed a significant reduction in oligomeric species of Abeta while immuno-staining of the 3(rd) instar larval brains showed a significant reduction in Abeta accumulation. Computational studies, as well as NMR and CD spectroscopy provide mechanistic insight into the activity of the compound which is most likely mediated by clamping of the aromatic recognition interface in the central segment of Abeta. Our results demonstrate that interfering with the aromatic core of amyloidogenic peptides is a promising approach for inhibiting various pathogenic species associated with amyloidogenic diseases. The compound NQTrp can serve as a lead for developing a new class of disease modifying drugs for Alzheimer's disease.