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Alzheimer's disease (AD) is a common neurodegenerative disease characterized by a neuroinflammatory state and to date, there is no cure and its treatment represents a large unmet clinical need. The involvement of T helper 17 cells in the pathogenesis of AD-related neuroinflammation has been reported in several studies, however the role of the main cytokine, IL-17, has not been well addressed. Herein, we investigate the effects of IL-17 neutralizing antibody (IL-17Ab) injected by intracerebroventricular (ICV) or intranasal (IN) routes on amyloid-β-induced neuroinflammation and memory impairment in mice.

L-cysteine is currently recognized as a conditionally essential sulphur amino acid. Besides contributing to many biological pathways, cysteine is a key component of the keratin protein by its ability to form disulfide bridges that confer strength and rigidity to the protein. In addition to cysteine, iron represents another critical factor in regulating keratins expression in epidermal tissues, as well as in hair follicle growth and maturation. By focusing on human keratinocytes, the aim of this study was to evaluate the effect of cysteine supplementation as nutraceutical on keratin biosynthesis, as well as to get an insight on the interplay of cysteine availability and cellular iron status in regulating keratins expression in vitro. Herein we demonstrate that cysteine promotes a significant up-regulation of keratins expression as a result of de novo protein synthesis, while the lack of iron impairs keratin expression. Interestingly, cysteine supplementation counteracts the adverse effect of iron deficiency on cellular keratin expression. This effect was likely mediated by the up-regulation of transferrin receptor and ferritin, the main cellular proteins involved in iron homeostasis, at last affecting the labile iron pool. In this manner, cysteine may also enhance the metabolic iron availability for DNA synthesis without creating a detrimental condition of iron overload. To the best of our knowledge, this is one of the first study in an in vitro keratinocyte model providing evidence that cysteine and iron cooperate for keratins expression, indicative of their central role in maintaining healthy epithelia. This article is protected by copyright. All rights reserved.

The formation of the complex cerebellar cortical circuits follows different phases, with initial synaptogenesis and subsequent processes of refinement guided by a variety of mechanisms. The regularity of the cellular and synaptic organization of the cerebellar cortex allowed detailed studies of the structural plasticity mechanisms underlying the formation of new synapses and retraction of redundant ones. For the attainment of the monoinnervation of the Purkinje cell by a single climbing fiber, several signals are involved, including electrical activity, contact signals, homosynaptic and heterosynaptic interaction, calcium transients, postsynaptic receptors, and transduction pathways. An important role in this developmental program is played by serotonergic projections that, acting on temporally and spatially regulated postsynaptic receptors, induce and modulate the phases of synaptic formation and maturation. In the adult cerebellar cortex, many developmental mechanisms persist but play different roles, such as supporting synaptic plasticity during learning and formation of cerebellar memory traces. A dysfunction at any stage of this process can lead to disorders of cerebellar origin, which include autism spectrum disorders but are not limited to motor deficits. Recent evidence in animal models links impairment of Purkinje cell function with autism-like symptoms including sociability deficits, stereotyped movements, and interspecific communication by vocalization.

The 5-HT7 receptor is the most recently identified member of the 5-HT receptor family. It is involved in the modulation of learning and memory processes and interacts with several signaling systems in the central nervous system including glutamate receptors. Since the cerebellum receives a rich serotoninergic innervation that originates in the reticular formation, we investigated the function and signaling properties of 5-HT7 receptors on the excitatory synapses received by Purkinje cells. We found that the activation of 5-HT7 by the selective agonist LP-211 (1 µM) produced an inhibitory effect on parallel fiber???evoked postsynaptic currents (EPSCs), that was prevented by the 5-HT7 receptor antagonist, SB-269970, but not by 5-HT1 receptor antagonist., WAY-100635. The decrease of glutamatergic response induced by LP was postsynaptically expressed, in accordance with the immunochemical localization of 5-HT7 at the Purkinje cell body and dendrites, and required the protein kinase A.EPSCs evoked by climbing fiber stimulation were not affected by LP. These results suggest the existence of a new mechanism of regulation allowing the serotonin afferents to refine the signals arriving to Purkinje cells during learning.

The ability to identify and avoid environmental stimuli that signal danger is essential to survival. Our understanding of how the brain encodes aversive behaviors has been primarily focused on roles for the amygdala, hippocampus (HIPP), prefrontal cortex, ventral midbrain, and ventral striatum. Relatively little attention has been paid to contributions from the dorsal striatum (DS) to aversive learning, despite its well-established role in stimulus-response learning. Here, we review studies exploring the role of DS in aversive learning, including different roles for the dorsomedial and dorsolateral striatum in Pavlovian fear conditioning as well as innate and inhibitory avoidance (IA) behaviors. We outline how future investigation might determine specific contributions from DS subregions, cell types, and connections that contribute to aversive behavior.

Numerous studies indicate that the cerebellum undergoes structural and functional neurodegenerative changes in Alzheimer's disease. The purpose of this study was to examine the extent of cerebellar alterations at early, preplaque stage of the pathology in TgCRND8 mice through behavioral, electrophysiological, and molecular analysis. Balance beam test and foot-printing analysis revealed significant motor coordination and balance deficits in 2-month-old TgCRND8 mice compared to their littermates. Patch-clamp recordings performed on cerebellar slices of transgenic mice showed synaptic plasticity deficit and loss of noradrenergic modulation at parallel fiber-Purkinje cell synapse suggesting an early dysfunction of the cerebellar circuitry due to amyloid precursor protein overexpression. Finally, western blot analysis revealed an enhanced expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits p47(phox) and p67(phox) as well as Ca2+/calmodulin-dependent protein kinase and protein kinase C alpha in the cerebellum of 2-month-old transgenic mice. Therefore, we propose the existence of self-sustaining feedback loop involving the formyl peptide receptor 2-reactive oxygen species-Ca2+/calmodulin-dependent protein kinase II-protein kinase C alpha pathway that may promote reactive oxygen species generation in the early stage of Alzheimer's disease and eventually contribute to the exacerbation of pathological phenotype.

Alzheimer's disease (AD) is a common form of dementia mainly characterized by the deposition of neurofibrillary tangles and β-amyloid (Aβ) peptides in the brain. Additionally, increasing evidence demonstrates that a neuro-inflammatory state plays a key role in the development of this disease. Beside synthetic drugs, the use of natural compounds represents an alternative for the development of new potential drugs for the treatment of AD. Among these, the root of Salvia miltiorhiza Bunge (also known as Danshen) used for the treatment of cardiovascular, cerebrovascular disease and CNS functional decline in Chinese traditional medicine is one of the most representative examples. We therefore evaluated the effects of tanshinone IIA (TIIA) and cryptotanshinone (CRY) (the two major lipophilic compounds of Danshen) in a non-genetic mouse model of β-amyloid (Aβ)-induced AD, which is mainly characterized by reactive gliosis and neuro-inflammation in the brain. To this aim, mice were injected intracerebroventricularly (i.c.v.) with Aβ1-42 peptide (3μg/3μl) and after with TIIA and CRY (1, 3, or 10mg/kg) intraperitoneally (i.p.) 3 times weekly for 21days following the induction of experimental AD. Spatial working memory was assessed as a measure of short-term memory in mice, whereas the level of GFAP, S100β, COX-2, iNOS and NF-kBp65 monitored by western blot and ELISA assay, were selected as markers of reactive gliosis and neuro-inflammation. Finally, by docking studies, the modulation of key pro-inflammatory enzymes and pathways involved in the AD-related neuro-inflammation were also investigated. Results indicate that TIIA and CRY alleviate memory decline in Aβ1-42-injected mice, in a dose dependent manner. Moreover, the analysis of gliosis-related and neuro-inflammatory markers in the hippocampal tissues reveal a remarkable reduction in the expression of GFAP, S100β, COX-2, iNOS and NF-kBp65 after CRY (10mg/kg) treatment. These effects were less evident, but still significant, after TIIA (10mg/kg). Finally, in silico analysis also revealed that both compounds were able to interact with the binding sites of NF-kBp65 endorsing the data from biochemical analysis. We conclude that TIIA and CRY display anti-inflammatory and neuroprotective effect in a non- genetic mouse model of AD, thus playing a role in slowing down the course and onset of AD.

The 5-HT7 receptor (5-HT7R) mediates important physiological effects of serotonin, such as memory and emotion, and is emerging as a therapeutic target for the treatment of cognitive disorders and depression. Although previous studies have revealed an expression of 5-HT7R in cerebellum, particularly at Purkinje cells, its functional role and signaling mechanisms have never been described. Using patch-clamp recordings in cerebellar slices of adult mice, we investigated the effects of a selective 5-HT7R agonist, LP-211, on the main plastic site of the cerebellar cortex, the parallel fiber-Purkinje cell synapse. Here we show that 5-HT7R activation induces long-term depression of parallel fiber-Purkinje cell synapse via a postsynaptic mechanism that involves the PKC-MAPK signaling pathway. Moreover, a 5-HT7R antagonist abolished the expression of PF-LTD, produced by pairing parallel fiber stimulation with Purkinje cell depolarization; whereas, application of a 5-HT7R agonist impaired LTP induced by 1 Hz parallel fiber stimulation. Our results indicate for the first time that 5-HT7R exerts a fine regulation of cerebellar bidirectional synaptic plasticity that might be involved in cognitive processes and neuropsychiatric disorders involving the cerebellum.

The role of the cerebellum in Alzheimer’s disease (AD) has been neglected for a long time. Recent studies carried out using transgenic mouse models have demonstrated that amyloid-β (Aβ) is deposited in the cerebellum and affects synaptic transmission and plasticity, sometimes before plaque formation. A wide variability of motor phenotype has been observed in the different murine models of AD, without a consistent correlation with the extent of cerebellar histopathological changes or with cognitive deficits. The loss of noradrenergic drive may contribute to the impairment of cerebellar synaptic function and motor learning observed in these mice. Furthermore, cerebellar neurons, particularly granule cells, have been used as in vitro model of Aβ-induced neuronal damage. An unexpected conclusion is that the cerebellum, for a long time thought to be somehow protected from AD pathology, is actually considered as a region vulnerable to Aβ toxic damage, even at the early stage of the disease, with consequences on motor performance.

The c-Jun N-terminal kinase (JNK) is part of a stress signalling pathway strongly activated by NMDA-stimulation and involved in synaptic plasticity. Many studies have been focused on the post-synaptic mechanism of JNK action, and less is known about JNK presynaptic localization and its physiological role at this site. Here we examined whether JNK is present at the presynaptic site and its activity after presynaptic NMDA receptors stimulation. By using N-SIM Structured Super Resolution Microscopy as well as biochemical approaches, we demonstrated that presynaptic fractions contained significant amount of JNK protein and its activated form. By means of modelling design, we found that JNK, via the JBD domain, acts as a physiological effector on T-SNARE proteins; then using biochemical approaches we demonstrated the interaction between Syntaxin-1-JNK, Syntaxin-2-JNK, and Snap25-JNK. In addition, taking advance of the specific JNK inhibitor peptide, D-JNKI1, we defined JNK action on the SNARE complex formation. Finally, electrophysiological recordings confirmed the role of JNK in the presynaptic modulation of vesicle release. These data suggest that JNK-dependent phosphorylation of T-SNARE proteins may have an important functional role in synaptic plasticity.

The formation of the complex cerebellar cortical circuits follows different phases, with initial synaptogenesis and subsequent processes of refinement guided by a variety of mechanisms. The regularity of the cellular and synaptic organization of the cerebellar cortex allowed detailed studies of the structural plasticity mechanisms underlying the formation of new synapses and retraction of redundant ones. For the attainment of the monoinnervation of the Purkinje cell by a single climbing fiber, several signals are involved, including electrical activity, contact signals, homosynaptic and heterosynaptic interaction, calcium transients, postsynaptic receptors, and transduction pathways. An important role in this developmental program is played by serotonergic projections that, acting on temporally and spatially regulated postsynaptic receptors, induce and modulate the phases of synaptic formation and maturation. In the adult cerebellar cortex, many developmental mechanisms persist but play different roles, such as supporting synaptic plasticity during learning and formation of cerebellar memory traces. A dysfunction at any stage of this process can lead to disorders of cerebellar origin, which include autism spectrum disorders but are not limited to motor deficits. Recent evidence in animal models links impairment of Purkinje cell function with autism-like symptoms including sociability deficits, stereotyped movements, and interspecific communication by vocalization.

Studies in vitro have demonstrated that β3-adrenergic receptors (β3-ARs) regulate protein metabolism in skeletal muscle by promoting protein synthesis and inhibiting protein degradation. In this study, we evaluated whether activation of β3-ARs by the selective agonist CL316,243 modifies the functional and structural properties of skeletal muscles of healthy mice. Daily injections of CL316,243 for 15 days resulted in a significant improvement in muscle force production, assessed by grip strength and weight tests and an increased myofiber cross-sectional area, indicative of muscle hypertrophy. In addition, atomic force microscopy revealed a significant effect of CL316,243 on the transversal stiffness of isolated muscle fibers. Interestingly, the expression level of mammalian target of rapamycin (mTOR) downstream targets and neuronal nitric oxide synthase (NOS) was also found to be enhanced in tibialis anterior and soleus muscles of CL316,243 treated mice, in accordance with previous data linking β3-ARs to mTOR and NOS signaling pathways. In conclusion, our data suggest that CL316,243 systemic administration might be a novel therapeutic strategy worthy of further investigations in conditions of muscle wasting and weakness associated with aging and muscular diseases.

The parallel fiber-Purkinje cell synapse represents the point of maximal signal divergence in the cerebellar cortex with an estimated number of about 60 billion synaptic contacts in the rat and 100,000 billions in humans. At the same time, the Purkinje cell dendritic tree is a site of remarkable convergence of more than 100,000 parallel fiber synapses. Parallel fibers activity generates fast postsynaptic currents via AMPA receptors, and slower signals, mediated by mGlu1 receptors, resulting in Purkinje cell depolarization accompanied by sharp calcium elevation within dendritic regions. Long-term depression and long-term potentiation have been widely described for the parallel fiber-Purkinje cell synapse and have been proposed as mechanisms for motor learning. The mechanisms of induction for LTP and LTD involve different signaling mechanisms within the presynaptic terminal and/or at the postsynaptic site, promoting enduring modification in the neurotransmitter release and change in responsiveness to the neurotransmitter. The parallel fiber-Purkinje cell synapse is finely modulated by several neurotransmitters, including serotonin, noradrenaline, and acetylcholine. The ability of these neuromodulators to gate LTP and LTD at the parallel fiber-Purkinje cell synapse could, at least in part, explain their effect on cerebellar-dependent learning and memory paradigms. Overall, these findings have important implications for understanding the cerebellar involvement in a series of pathological conditions, ranging from ataxia to autism. For example, parallel fiber-Purkinje cell synapse dysfunctions have been identified in several murine models of spinocerebellar ataxia (SCA) types 1, 3, 5 and 27. In some cases, the defect is specific for the AMPA receptor signaling (SCA27), while in others the mGlu1 pathway is affected (SCA1, 3, 5). Interestingly, the parallel fiber-Purkinje cell synapse has been shown to be hyper-functional in a mutant mouse model of autism spectrum disorder, with a selective deletion of Pten in Purkinje cells. However, the full range of methodological approaches, that allowed the discovery of the physiological principles of PF-PC synapse function, has not yet been completely exploited to investigate the pathophysiological mechanisms of diseases involving the cerebellum. We, therefore, propose to extend the spectrum of experimental investigations to tackle this problem.

To examine how signals from different sensory modalities are integrated to generate an appropriate goal-oriented behavior, we trained rats in an eight-arm radial maze to visit a cue arm provided with intramaze cues from different sensory modalities, i.e. visual, tactile and auditory, in order to obtain a reward. When the same rats were then examined on test trials in which the cue arm contained one of the stimuli that the animals were trained with (i.e. light, sound or rough sheet), they showed a significant impairment with respect to the performance on the polymodal-cue task. The contribution of the dorsal hippocampus to the acquisition and retention of polymodal-cue guided task was also examined. We found that rats with dorsal hippocampal lesions before training showed a significant deficit in the acquisition of polymodal-cue oriented task that improved with overtraining. The selective lesion of the dorsal hippocampus after training disrupted memory retention, but the animals' performance improved following retraining of the polymodal task. All hippocampal lesioned rats displayed an impaired performance on the unimodal test. These findings suggest that the dorsal hippocampus contributes to the processing of multimodal sensory information for the associative memory formation and consolidation.

The signals arriving to Purkinje cells via parallel fibers are essential for all tasks in which the cerebellum is involved, including motor control, learning new motor skills and calibration of reflexes. Since learning also requires the activation of adrenergic receptors, we investigated the effects of adrenergic receptor agonists on the main plastic site of the cerebellar cortex, the parallel fiber-Purkinje cell synapse. Here we show that noradrenaline serves as an endogenous ligand for both α1-and α2-adrenergic receptors to produce synaptic depression between parallel fibers and Purkinje cells. On the contrary, PF-EPSCs were potentiated by the β-adrenergic receptor agonist isoproterenol. This short-term potentiation was postsynaptically expressed, required protein kinase A, and was mimicked by the β2-adrenoceptor agonist clenbuterol, suggesting that the β2-adrenoceptors mediate the noradrenergic facilitation of synaptic transmission between parallel fibers and Purkinje cells. Moreover, β-adrenoceptor activation lowered the threshold for cerebellar long-term potentiation induced by 1 Hz parallel fiber stimulation. The presence of both α and β adrenergic receptors on Purkinje cells suggests the existence of bidirectional mechanisms of regulation allowing the noradrenergic afferents to refine the signals arriving to Purkinje cells at particular arousal states or during learning.

With the help of single-cell microflorimetry, (45)Ca(2+) radiotracer fluxes, and patch-clamp in whole-cell configuration, we examined the effect of the amiloride derivative 3-amino-6-chloro-5-[(4-chloro-benzyl)amino]-N-[[(2,4-dimethylbenzyl)amino]iminomethyl]-pyrazinecarboxamide (CB-DMB) on the activity of the three isoforms of the Na(+)/Ca(2+) exchanger (NCX) and on several other membrane currents including voltage- and pH-sensitive ones. This amiloride analog suppressed the bidirectional activity of all NCX isoforms in a concentration-dependent manner. The IC(50) values of CB-DMB were in the nanomolar range for the outward and the inward components of the bidirectional NCX1, NCX2, and NCX3 activity. Deletion mutagenesis showed that CB-DMB inhibited NCX activity mainly at level of the f-loop but not through the interaction with Gly833 located at the level of the alpha(2) repeat. On the other hand, CB-DMB suppressed in the micromolar range the other plasma membrane currents encoded by voltage-dependent Ca(2+) channels, tetrodotoxin-sensitive Na(+) channels, and pH-sensitive ASIC1a. Collectively, the data of the present study showed that CB-DMB, when used in the nanomolar range, is one of the most potent compounds that can block the activity of the three NCX isoforms when they work both in the forward and in the reverse modes of operation without interfering with other ionic channels.