Do diencephalic sleep-wake-regulatory systems meet?

A wealth of classical studies on the thalamus pointed out a key role of the reticular thalamic nucleus (Rt) in sleep regulation. Since the discovery of the orexin (Orx)/ hypocretin peptides in 1998, a key role in wakefulness stability has been ascribed to Orx neurons, which reside in the lateral hypothalamus. Rt neuron efferents, which are inhibitory, are distributed to nuclei of the dorsal thalamus. Orx neuron efferents, which are excitatory, are distributed widely in the neuroaxis but concentrated in the thalamus along the midline. Current views seem to consider these systems as separate networks. We here wondered whether these networks meet instead in the diencephalon, and in particular whether Rt and Orx synaptic endings converge on the same neuronal cell bodies or reach separate neurons of the paraventricular thalamic nucleus (PVT) of the thalamic midline. To answer this question, PVT neurons were here investigated in confocal microscopy by means of multiple immunofluorescent labelling: calretinin labelling of PVT cell bodies; Orx + synaptophysin for the labelling of Orx synaptic endings; parvalbumin + synaptophysin for the labelling of Rt synaptic endings. Striking results on the convergence of the two sets of synapses on the same neurons were obtained, since almost 100% of Orx synaptic boutons were apposed to PVT neurons which also received Rt synaptic boutons. Rt axon terminals were more abundant in PVT than Orx ones, and also reached neurons which did not receive Orx input. The present findings on the synaptic wiring of PVT neurons therefore points to the dorsal thalamic midline as a “meeting point” of sleep-wake-regulatory diencephalic networks. PVT efferents reach the prefrontal cortex, and limbic targets represented by the nucleus accumbens and amygdala. The synaptic convergence here demonstrated could thus place PVT neurons at the core of sleep-wake-related modulation of cognitive functions and emotional, affective behaviour

Peptidergic innervation of the olfactory bulb: a sleep/wake-regulatory route through the nose

Olfactory epithelium receptor neurons in the nasal cavity, which are exposed to the external environment, reach the olfactory bulb (OB), representing a direct port of entry to the brain. Through retrograde axonal transport, pathogens, toxins and misfolded proteins can reach brain cell groups which innervate the OB and result in functional alterations. Indeed, influenza virus nasal instillation was found to target brainstem and hypothalamic cell groups and result in narcoleptic-like sleep/wake changes [1]. These cell groups included the wake-promoting orexin (OX)-containing neurons, and the sleep-promoting melanin-concentrating hormone (MCH)-containing neurons [1]. Orexinergic innervation of the OB has been reported, but OX and MCH neurons innervating the OB have never been visualized. OX immunoreactivity in the mouse olfactory receptor neurons has been ascribed to the olfactory mucosa. Sources of input to the OB have been studied [2] before the discovery of OX in 1998. Orexinergic innervation of the prefrontal cortex is instead well established. Aim of this study was to reveal OX- and MCH-containing neurons projecting to the OB. Unilateral injections of the retrograde fluorescent tracer Fluoro-Gold (FG) confined to the OB of adult mice were combined with immunophenotyping and quantitative analysis of retrogradely labeled neurons. The findings were compared with those obtained after FG injections in the prefrontal cortex. Following FG injections in the OB, labeled neurons were found in the ipsilateral lateral hypothalamus, and included intermingled OX-A- or MCH-immunoreactive cells. About 8% of orexinergic neurons were labeled when the tracer was confined to the OB. This proportion increased (13±2.49 %) in cases in which a faint halo of tracer diffusion to the lateral portion of the prefrontal cortex was observed. Preliminary data indicate retrograde labeling from the OB of almost 15% of MCH-containing neurons. The findings demonstrate that OX and MCH neurons reach the OB directly, thus providing to environmental agents a route to sleep/wake-regulatory nodes via the nasal cavity

Converging orexinergic and reticular thalamic inputs on thalamic paraventricular neurons in normal conditions and experimental sleeping sickness

A subset of excitatory neurons in the lateral hypothalamus, known to express the peptide orexin/hypocretin (Ox), play a key role in maintaining wakefulness. Projections from Ox neurons are widely distributed in the neuraxis but terminations are heavily concentrated in the thalamus along the midline, especially the paraventricular thalamic nucleus (PVT). The same areas receive afferents from inhibitory, GABAergic neurons expressing parvalbumin (Pv) in the thalamic reticular nucleus (Rt), which has long been considered essential for sleep regulation. While the two circuitries have been regarded as distinct, we tested the hypothesis that PVT neurons represent a common target for both afferent systems by means of confocal microscopy of multiple immunofluorescence labeling in the mouse brain. Calretinin (Cr) was used as marker of PVT neurons. Almost 90% of PVT perikarya were contacted by Pv+ terminals, confirming the prominent role of Rt in modulating PVT activity. Interestingly, about a third of these neurons were also reached by Ox+ terminals, suggesting a key role of the thalamic midline in integrating information pertaining vigilance state control. PVT cells receiving Ox+ but not Pv+ contacts were observed only rarely. In mice infected with the parasite Trypanosoma brucei brucei, the causal agent of the neuroinflammatory disease “sleeping sickness”, Pv+ afferents into PVT were largely preserved, while orexinergic fibers appeared fragmented and reduced in density. Importantly, the fraction of PVT perikarya receiving both Pv+ and Ox+ terminals was reduced by about 50%. The substantially decreased convergence of the two regulatory systems, in association with infection-induced disrupted sleep and sleep/wake cycles, further supports the hypothesis that PVT contributes to vigilance and arousal in physiological conditions

BID and the α-bisabolol-triggered cell death program: converging on mitochondria and lysosomes

\u3b1-Bisabolol (BSB) is a plant-derived sesquiterpene alcohol able to trigger regulated cell death in transformed cells, while deprived of the general toxicity in several mouse models. Here, we investigated the involvement of lysosomal and mitochondrial compartments in the cytotoxic effects of BSB, with a specific focus on the BH3-only activator protein BID. We found that BSB particularly accumulated in cancer cell lines, displaying a higher amount of lipid rafts as compared to normal blood cells. By means of western blotting and microscopy techniques, we documented rapid BSB-induced BID translocation to lysosomes and mitochondria, both of them becoming dysfunctional. Lysosomal membranes were permeabilized, thus blocking the cytoprotective autophagic flux and provoking cathepsin B leakage into the cytosol. Multiple flow cytometry-based experiments demonstrated the loss of mitochondrial membrane potential due to pore formation across the lipid bilayer. These parallel events converged on neoplastic cell death, an outcome significantly prevented by BID knockdown. Therefore, BSB promoted BID redistribution to the cell death executioner organelles, which in turn activated anti-autophagic and proapoptotic mechanisms. This is an example of how xenohormesis can be exploited to modulate basic cellular programs in cancer

A new monoclonal antibody detects downregulation of protein tyrosine phosphatase receptor type γ in chronic myeloid leukemia patients

Background: Protein tyrosine phosphatase receptor gamma (PTPRG) is a ubiquitously expressed member of the protein tyrosine phosphatase family known to act as a tumor suppressor gene in many different neoplasms with mechanisms of inactivation including mutations and methylation of CpG islands in the promoter region. Although a critical role in human hematopoiesis and an oncosuppressor role in chronic myeloid leukemia (CML) have been reported, only one polyclonal antibody (named chPTPRG) has been described as capable of recognizing the native antigen of this phosphatase by flow cytometry. Protein biomarkers of CML have not yet found applications in the clinic, and in this study, we have analyzed a group of newly diagnosed CML patients before and after treatment. The aim of this work was to characterize and exploit a newly developed murine monoclonal antibody specific for the PTPRG extracellular domain (named TPγ B9-2) to better define PTPRG protein downregulation in CML patients. Methods: TPγ B9-2 specifically recognizes PTPRG (both human and murine) by flow cytometry, western blotting, immunoprecipitation, and immunohistochemistry. Results: Co-localization experiments performed with both anti-PTPRG antibodies identified the presence of isoforms and confirmed protein downregulation at diagnosis in the Philadelphia-positive myeloid lineage (including CD34+/CD38bright/dim cells). After effective tyrosine kinase inhibitor (TKI) treatment, its expression recovered in tandem with the return of Philadelphia-negative hematopoiesis. Of note, PTPRG mRNA levels remain unchanged in tyrosine kinase inhibitors (TKI) non-responder patients, confirming that downregulation selectively occurs in primary CML cells. Conclusions: The availability of this unique antibody permits its evaluation for clinical application including the support for diagnosis and follow-up of these disorders. Evaluation of PTPRG as a potential therapeutic target is also facilitated by the availability of a specific reagent capable to specifically detect its target in various experimental conditions

Therapeutic targeting of Lyn kinase to treat chorea-acanthocytosis

Chorea-Acanthocytosis (ChAc) is a devastating, little understood, and currently untreatable neurodegenerative disease caused by VPS13A mutations. Based on our recent demonstration that accumulation of activated Lyn tyrosine kinase is a key pathophysiological event in human ChAc cells, we took advantage of Vps13a-/- mice, which phenocopied human ChAc. Using proteomic approach, we found accumulation of active Lyn, \u3b3-synuclein and phospho-tau proteins in Vps13a-/- basal ganglia secondary to impaired autophagy leading to neuroinflammation. Mice double knockout Vps13a-/- Lyn-/- showed normalization of red cell morphology and improvement of autophagy in basal ganglia. We then in vivo tested pharmacologic inhibitors of Lyn: dasatinib and nilotinib. Dasatinib failed to cross the mouse brain blood barrier (BBB), but the more specific Lyn kinase inhibitor nilotinib, crosses the BBB. Nilotinib ameliorates both Vps13a-/- hematological and neurological phenotypes, improving autophagy and preventing neuroinflammation. Our data support the proposal to repurpose nilotinib as new therapeutic option for ChAc patients

Synaptic competition in central and peripheral nervous system: role of the postsynaptic cell

Non disponibileIn central and peripheral nervous system the formation of a precise pattern of connections between neurons is the result of competitive synaptic processes occurring during development. In the embryonic stage neurons from redundant connetcions with a higher amount of target cells respect to those present in the adult. After birth supernumerary connections are lost after a process of peripheral refinement in which different inputs complete for the same target. Such process, called synaptic elimination, is generally accepted to be activity-dependent. The study of these competitive phenomena is considerably simplified in models in which at the end of the synaptic elimination the target cell is innervated by only one axon. These systems pass from a pattern of polyneuronal innervation to monoinnervation. In this PhD thesis two of these models are treated in different sections: the cerebellar cortex and the neuromuscolar junction. During development, in the cerebellar cortex various climbing fibers (CFs) innervate the perisomatic portion of a single Purkinje cell (PC). After a synaptic competition and elimination processes occurring mainly in the second postnatal week in mice and rats, each PC retains only one CF. In parallel climbing fibers terminations leaves the PC soma and are translocated in proximal dendritic compartment

Sos1 regulates macrophage podosome assembly and macrophage invasive capacity

Podosomes are protrusive structures implicated in macrophage extracellular matrix degradation and three-dimensional migration through cell barriers and the interstitium. Podosome formation and assembly are regulated by cytoskeleton remodeling requiring cytoplasmic tyrosine kinases of the Src and the Abl families. Considering that Abl has been reported to phosphorylate the guanine nucleotide exchange factor Sos1, eliciting its Rac-guanine nucleotide exchange factor activity, and Rac regulates podosome formation in myeloid cells and invadopodia formation in cancer cells, we addressed whether Sos1 is implicated in podosome formation and function in macrophages. We found that ectopically expressed Abl or the Src kinase Fgr phosphorylate Sos1, and the Src kinases Hck and Fgr are required for Abl and Sos1 phosphorylation and Abl/Sos1 interaction in macrophages. Sos1 localizes to podosomes in both murine and human macrophages, and its silencing by small interfering RNA results in disassembly of murine macrophage podosomes and a marked reduction of GTP loading on Rac. Matrix degradative capacity, three-dimensional migration through Matrigel, and transmigration through an endothelial cell monolayer of Sos1-silenced macrophages were inhibited. In addition, Sos1- or Abl-silenced macrophages, or macrophages treated with the selective Abl inhibitor imatinib mesylate had a reduced capability to migrate into breast tumor spheroids, the majority of cells remaining at the margin and the outer layers of the spheroid itself. Because of the established role of Src and Abl kinases to regulate also invadopodia formation in cancer cells, our findings suggest that targeting the Src/Abl/Sos1/Rac pathway may represent a double-edged sword to control both cancer-invasive capacities and cancer-related inflammation

Synapse formation and elimination: role of activity studied in different models of adult muscle reinnervation.

Synapse competition and elimination are a general developmental process both in central and in peripheral nervous systems that is strongly activity dependent. Some common features regulate synapse competition, and one of these is an application to development of the Hebb's postulate of learning: repeated coincident spike activity in competing presynaptic inputs on the same target cell inhibits competition, whereas noncoincident activity promotes weakening of some of the inputs and ultimately their elimination. Here we report experiments that indicate that the development of muscle innervation (initial polyneuronal innervation and subsequent synapse elimination) follows the Hebb's paradigm. We utilized two different models of muscle reinnervation in the adult rat: 1) we crushed nerves going to soleus or extensor digitorum longus muscles, to activate regeneration of the presynaptic component of the neuromuscular junctions (NMJ), or 2) we injected the soleus muscle with Marcaine (a myotoxic agent) to activate regeneration of the postsynaptic component, the muscle fiber. A condition of transient polyneuronal innervation occurs during NMJ regeneration in both cases, although the two models differ insofar as the relative strength of the competing inputs is concerned. During the period of competition (a few days or weeks, in Marcaine or crush experiments, respectively), we imposed a synchronous firing pattern on the competing inputs by stimulating motor axons distal to a chronic conduction block and demonstrated that this procedure strongly inhibits synapse elimination, with respect to control muscles in which regeneration occurs under natural impulse activity of motoneurons