The manipulation of endogenous stem cell populations from the subventricular zone (SVZ), a neurogenic niche, creates an opportunity to induce neurogenesis and influence brain regenerative capacities in the adult brain. Herein, we demonstrate the ability of polyelectrolyte nanoparticles to induce neurogenesis exclusively after being internalized by SVZ stem cells. The nanoparticles are not cytotoxic for concentrations equal or below 10 μg/mL. The internalization process is rapid, and nanoparticles escape endosomal fate in a few hours. Retinoic acid-loaded nanoparticles increase the number of neuronal nuclear protein (NeuN)-positive neurons and functional neurons responding to depolarization with KCl and expressing NMDA receptor subunit type 1 (NR1). These nanoparticles offer an opportunity for in vivo delivery of proneurogenic factors and neurodegenerative disease treatment.

Pollen grains with distinct allergenic abilities release proteases that might be involved in the sensitization to a range of airborne allergens by facilitating allergen delivery across the epithelium and also contribute directly to the inflammation characteristic of allergic diseases.

Machado-Joseph's disease is caused by a CAG trinucleotide repeat expansion that is translated into an abnormally long polyglutamine tract in the protein ataxin-3. Except for the polyglutamine region, proteins associated with polyglutamine diseases are unrelated, and for all of these diseases aggregates containing these proteins are the major components of the nuclear proteinaceous deposits found in the brain. Aggregates of the expanded proteins display amyloid-like morphological and biophysical properties. Human ataxin-3 containing a non-pathological number of glutamine residues (14Q), as well as its Caenorhabditis elegans (1Q) orthologue, showed a high tendency towards self-interaction and aggregation, under near-physiological conditions. In order to understand the discrete steps in the assembly process leading to ataxin-3 oligomerization, we have separated chromatographically high molecular mass oligomers as well as medium mass multimers of non-expanded ataxin-3. We show that: (a) oligomerization occurs independently of the poly(Q)-repeat and it is accompanied by an increase in beta-structure; and (b) the first intermediate in the oligomerization pathway is a Josephin domain-mediated dimer of ataxin-3. Furthermore, non-expanded ataxin-3 oligomers are recognized by a specific antibody that targets a conformational epitope present in soluble cytotoxic species found in the fibrillization pathway of expanded polyglutamine proteins and other amyloid-forming proteins. Imaging of the oligomeric forms of the non-pathological protein using electron microscopy reveals globular particles, as well as short chains of such particles that likely mimic the initial stages in the fibrillogenesis pathway occurring in the polyglutamine-expanded protein. Thus, they constitute potential targets for therapeutic approaches in Machado-Joseph's disease, as well as valuable diagnostic markers in disease settings.

Brain inflammation is characterized primarily by microglia activation (1). Several stimuli, such as ATP (2), bloodderived factors, or microbial signals (e.g. lipopolysaccharide (LPS)), induce significant morphological changes in microglial cells (3). They become undistinguishable from active macrophages and are able to migrate and proliferate at sites of neuronal injury, where they release both neurotrophic and neurotoxic factors, and inflammatory mediators, such as adhesion molecules, cytokines, and complement molecules among others (4 -6). Consequently, microglia response remains controversial because it can either be beneficial or deleterious depending on the nature, concentration, and time of exposure to the activating stimulus, and the cellular interactions of the targeted tissue. Once the triggering stimulus wanes, microglia participate in the down-modulation of the immune response and in the regulation of their own apoptosis via secretion of antiinflammatory cytokines (3).One of the outcomes of microglia activation is the production of nitric oxide (NO) from the conversion of L-arginine to L-citrulline by Ca 2ϩ -independent inducible nitric oxide synthase (iNOS) 2 (7-9). NO is produced by numerous cells, and it is of particular importance for blood flow regulation, sleepwake cycle, food intake and thermal regulation, immune system function, and neuronal transmission (10). Particularly, in the central nervous system, NO regulation presents itself as an opportunity to intervene in human health. NO can grant neuroprotection through the following mechanisms: reduction of Ca 2ϩ influx, due to S-nitrosylation of caspase 3 and NR1 and NR2 subunits of the N-methyl-D-aspartate receptors, which leads to a decrease of cell death; activation of cyclic AMP-responsive element-binding protein and Akt via stimulation of the soluble guanylate cyclase-cyclic GMP-protein kinase G pathway; and generation of biliverdin, a precursor of bilirubin, which acts as an antioxidant and antinitrosive molecule, through the induction of the activity of heme oxygenase 1 (10).However, NO can act as a pathophysiological agent because it is associated with hypertension, diabetes, and heart failure among other pathologies (8). In the central nervous system, high amounts of NO inhibit mitochondrial cytochrome oxidase in neurons, causing them to depolarize and to release glutamate and ultimately to die by excitotoxicity via N-methyl-D-aspartate receptors (11,12). NO can also react with superoxide anions and form peroxynitrite, which detains strong oxidant properties and can damage cellular components when protein nitration takes place (10).

Plant aspartic proteinases contain a plant-specific insert (PSI) of about 100 amino acids of unknown function with no similarity with the other aspartic proteinases but with significant similarity with saposins, animal sphingolipid activator proteins. PSI has remained elusive at the protein level, suggesting that it may be removed during processing. To understand the molecular relevance of PSI, the proteolytic processing of cardosin A, the major aspartic proteinase from the flowers of cardoon (Cynara cardunculus L.) was studied. Procardosin A, a 64-kDa cardosin A precursor containing PSI and the prosegment was identified by immunoblotting using monospecific antibodies against PSI and the prosegment. Procardosin A undergoes proteolytic processing as the flower matures. PSI was found to be removed before the prosegment, indicating that during processing the enzyme acquires a structure typical of mammalian or microbial aspartic proteinase proforms. In vitro studies showed that processing of PSI occurs at pH 3.0 and is inhibited by pepstatin A and at pH 7.0. Sequence analysis allowed the identification of the cleavage sites, revealing that PSI is removed entirely, probably by an aspartic proteinase. Cleavage of the PSI scissile bonds requires, however, a conformation specific to the precursor since isolated cardosins and pistil extracts were unable to hydrolyse synthetic peptides corresponding to the cleavage sites. In view of these results, a model for the proteolytic processing of cardosin A is proposed and the molecular and physiological relevance of PSI in plant aspartic proteinase is discussed.Keywords : aspartic protease ; milk-clotting enzyme; cardosin ; proteolytic processing; saposin.Aspartic proteinases (AP) are a widely distributed class of kingdom [5]. In the majority of plants AP are located in seeds, endoproteases that share significant similarities at the amino-whether quiescent or germinating. They are believed to particiacid-sequence level and at the structural level [1, 2]. The typical pate in storage-protein cleavage, which is necessary for germinacharacteristics of the family are an acidic pH optimum, inhibi-tion [5,10]. AP have also been found in leaves of some plants. tion by pepstatin A, preference for bonds between hydrophobic In leaves they have been implicated in mechanisms of defense amino acids, and sequence similarities, in particular the conser-against pathogens [11,12]. Species of the genus Cynara contain vation of the catalytic triads Asp-Thr-Gly or Asp-Ser-Gly [1Ϫ considerable amounts of AP in flowers. Recent data indicate that 4]. AP have been implicated in diverse physiological processes, the major AP from cardoon (Cynara cardunculus L.), cardosin such as digestion or blood-pressure regulation, and in some A, may be involved in the sexual reproduction of the plant [6] pathological conditions, including infection by fungi and retro-and may have a defensive role as well [13]. viruses or cancer [5, 6].A few primary structures deduced from the cDNAs of AP Like many other proteases, A...

Herein, we report the use of retinoic acid-loaded polymeric nanoparticles as a potent tool to induce the neuronal differentiation of subventricular zone neural stem cells. The intracellular delivery of retinoic acid by the nanoparticles activated nuclear retinoic acid receptors, decreased stemness, and increased proneurogenic gene expression. Importantly, this work reports for the first time a nanoparticle formulation able to modulate in vivo the subventricular zone neurogenic niche. The work further compares the dynamics of initial stages of differentiation between SVZ cells treated with retinoic acid-loaded polymeric nanoparticles and solubilized retinoic acid. The nanoparticle formulation developed here may ultimately offer new perspectives to treat neurodegenerative diseases.

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J. Neurochem. (2012) 120, 93–105. Abstract Increasing evidences suggest that neuropeptide Y (NPY) may act as a key modulator of the cross‐talk between the brain and the immune system in health and disease. In the present study, we dissected the possible inhibitory role of NPY upon inflammation‐associated microglial cell motility. NPY, through activation of Y1 receptors, was found to inhibit lipopolysaccharide (LPS)‐induced microglia (N9 cell line) motility. Moreover, stimulation of microglia with LPS was inhibited by IL‐1 receptor antagonist (IL‐1ra), suggesting the involvement of endogenous interleukin‐1 beta (IL‐1β) in this process. Direct stimulation with IL‐1β promoted downstream p38 mitogen‐activated protein kinase mobilization and increased microglia motility. Moreover, consistently, p38 mitogen‐activated protein kinase inhibition decreased the extent of actin filament reorganization occurring during plasma membrane ruffling and p38 phosphorylation was inhibited by NPY, involving Y1 receptors. Significantly, the key inhibitory role of NPY on LPS‐induced motility of CD11b‐positive cells was further confirmed in mouse brain cortex explants. In summary, we revealed a novel functional role for NPY in the regulation of microglial function that may have important implications in the modulation of CNS injuries/diseases where microglia migration/motility might play a role.