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Several works reported increased differentiation of neuronal cells grown on graphene; however, the molecular mechanism driving axon elongation on this material has remained elusive. Here, we study the axonal transport of nerve growth factor (NGF), the neurotrophin supporting development of peripheral neurons, as a key player in the time course of axonal elongation of dorsal root ganglion neurons on graphene. We find that graphene drastically reduces the number of retrogradely transported NGF vesicles in favor of a stalled population in the first two days of culture, in which the boost of axon elongation is observed. This correlates with a mutual charge redistribution, observed via Raman spectroscopy and electrophysiological recordings. Furthermore, ultrastructural analysis indicates a reduced microtubule distance and an elongated axonal topology. Thus, both electrophysiological and structural effects can account for graphene action on neuron development. Unraveling the molecular players underneath this interplay may open new avenues for axon regeneration applications. Several works reported increased differentiation of neuronal cells grown on graphene; however, the molecular mechanism driving axon elongation on this material has remained elusive. Here, we study the axonal transport of nerve growth factor (NGF), the neurotrophin supporting development of peripheral neurons, as a key player in the time course of axonal elongation of dorsal root ganglion neurons on graphene. We find that graphene drastically reduces the number of retrogradely transported NGF vesicles in favor of a stalled population in the first 2 days of culture, in which the boost of axon elongation is observed. This correlates with a mutual charge redistribution, observed via Raman spectroscopy and electrophysiological recordings. Furthermore, ultrastructural analysis indicates a reduced microtubule distance and an elongated axonal topology. Thus, both electrophysiological and structural effects can account for graphene action on neuron development. Unraveling the molecular players underneath this interplay may open new avenues for axon regeneration applications.

Tau displacement from microtubules is the first step in the onset of tauopathies and is followed by toxic protein aggregation. However, other non-canonical functions of Tau might have a role in these pathologies. Here, we demonstrate that a small amount of Tau localizes in the nuclear compartment and accumulates in both the soluble and chromatin-bound fractions. We show that favoring Tau nuclear translocation and accumulation, by Tau overexpression or detachment from MTs, increases the expression of VGluT1, a disease-relevant gene directly involved in glutamatergic synaptic transmission. Remarkably, the P301L mutation, related to frontotemporal dementia FTDP-17, impairs this mechanism leading to a loss of function. Altogether, our results provide the demonstration of a direct physiological role of Tau on gene expression. Alterations of this mechanism may be at the basis of the onset of neurodegeneration.

The p75 neurotrophin (NT) receptor (p75(NTR)) plays a crucial role in balancing survival-versus-death decisions in the nervous system. Yet, despite 2 decades of structural and biochemical studies, a comprehensive, accepted model for p75(NTR) activation by NT ligands is still missing. Here, we present a single-molecule study of membrane p75(NTR) in living cells, demonstrating that the vast majority of receptors are monomers before and after NT activation. Interestingly, the stoichiometry and diffusion properties of the wild-type (wt) p75(NTR) are almost identical to those of a receptor mutant lacking residues previously believed to induce oligomerization. The wt p75(NTR) and mutated (mut) p75(NTR) differ in their partitioning in cholesterol-rich membrane regions upon nerve growth factor (NGF) stimulation: We argue that this is the origin of the ability of wt p75(NTR), but not of mut p75(NTR), to mediate immature NT (proNT)-induced apoptosis. Both p75(NTR) forms support proNT-induced growth cone retraction: We show that receptor surface accumulation is the driving force for cone collapse. Overall, our data unveil the multi-faceted activity of the p75(NTR) monomer and let us provide a coherent interpretative frame of existing conflicting data in the literature.

The Forkhead Box G1 (FOXG1) gene encodes a transcription factor with an essential role in mammalian telencephalon development. FOXG1-related disorders, caused by deletions, intragenic mutations or duplications, are usually associated with severe intellectual disability, autistic features, and, in 87% of subjects, epileptiform manifestations. In a subset of patients with FoxG1 mutations, seizures remain intractable, prompting the need for novel therapeutic options. To address this issue, we took advantage of a haploinsufficient animal model, the FoxG1 +/− mouse. In vivo electrophysiological analyses of FoxG1 +/− mice detected hippocampal hyperexcitability, which turned into overt seizures upon delivery of the proconvulsant kainic acid, as confirmed by behavioral observations. These alterations were associated with decreased expression of the chloride transporter KCC2. Next, we tested whether a triheptanoin-based anaplerotic diet could have an impact on the pathological phenotype of FoxG1 +/− mice. This manipulation abated altered neural activity and normalized enhanced susceptibility to proconvulsant-induced seizures, in addition to rescuing altered expression of KCC2 and increasing the levels of the GABA transporter vGAT. In conclusion, our data show that FoxG1 haploinsufficiency causes dysfunction of hippocampal circuits and increases the susceptibility to a proconvulsant insult, and that these alterations are rescued by triheptanoin dietary treatment.

Maternal obesity negatively affects fetal development. Abnormalities in brain glucose metabolism are predictive of metabolic-cognitive disorders.We studied the offspring (aged 0, 1, 6, 12 months) of minipigs fed a normal vs high-fat diet (HFD), by positron emission tomography (PET) to measure brain glucose metabolism, and ex vivo assessments of brain insulin receptors (IRβ) and GLUT4.At birth, brain glucose metabolism and IRβ were twice as high in the offspring of HFD-fed than control mothers. During infancy and youth, brain glucose uptake, GLUT4 and IRβ increased in the offspring of control mothers and decreased in those of HFD-fed mothers, leading to a 40-85% difference (p 0.05), and severe glycogen depletion, lasting until adulthood.Maternal high-fat feeding leads to brain glucose overexposure during fetal development, followed by long-lasting depression in brain glucose metabolism in minipigs. These features may predispose the offspring to develop metabolic-neurodegenerative diseases.

Leptin and Brain Derived Neurotrophic Factor (BDNF) pathways are critical players in body weight homeostasis. Noninvasive treatments like environmental stimulation are able to increase response to leptin and induce BDNF expression in the brain. Emerging evidences point to the antidepressant selective serotonin reuptake inhibitor Fluoxetine (FLX) as a drug with effects similar to environmental stimulation. FLX is known to impact on body weight, with mechanisms yet to be elucidated. We herein asked whether FLX affects energy balance, the leptin system and BDNF function. Adult lean male mice chronically treated with FLX showed reduced weight gain, higher energy expenditure, increased sensitivity to acute leptin, increased hypothalamic BDNF expression, associated to changes in white adipose tissue expression typical of “brownization”. In the Ntrk2tm1Ddg/J model, carrying a mutation in the BDNF receptor Tyrosine kinase B (TrkB), these effects are partially or totally reversed. Wild type obese mice treated with FLX showed reduced weight gain, increased energy output, and differently from untreated obese mice, a preserved acute response to leptin in terms of activation of the intracellular leptin transducer STAT3. In conclusion, FLX impacts on energy balance and induces leptin sensitivity and an intact TrkB function is required for these effects to take place.

Hippocampal plasticity is triggered by a variety of stimuli including sensory inputs, neurotrophins and inflammation. Leptin, whose primary function is to regulate food intake and energy expenditure, has been recently shown to affect hippocampal neurogenesis and plasticity. Interestingly, mice fed a high-fat diet (HFD) exhibit impaired hippocampal function, but the underlying mechanisms are poorly understood. To address this issue, we compared leptin responsiveness of hippocampal neurons in control and HFD-fed mice by combining single-cell electrophysiology and biochemical assays. We found that leptin modulated spontaneous and evoked synaptic transmission in control, but not HFD, mice. This functional impairment was paralleled by blunted activation of STAT-3, one of the key signal transduction pathways controlled by the fully functional isoform of the leptin receptor, ObRb. In addition, SOCS-3 expression was non-responsive to leptin, indicating that modulation of negative feedback impinging on ObRb was also altered. Our results advance the understanding of leptin action on hippocampal plasticity and, more importantly, suggest that leptin resistance is a key determinant of hippocampal dysfunction associated with hypercaloric diet.

Besides its classical function of bone metabolism regulation, 1alpha, 25-dihydroxyvitamin D3 (1,25(OH) 2 D 3 ), acts on a variety of tissues including the nervous system, where the hormone plays an important role as neuroprotective, antiproliferating and differentiating agent. Sphingolipids are bioactive lipids that play critical and complex roles in regulating cell fate. In the present paper we have investigated whether sphingolipids are involved in the protective action of 1,25(OH) 2 D 3. We have found that 1,25(OH) 2 D 3 prevents amyloid-β peptide (Aβ(1−42)) cytotoxicity both in differentiated SH-SY5Y human neuroblastoma cells and in vivo . In differentiated SH-SY5Y cells, Aβ(1−42) strongly reduces the sphingosine-1-phosphate (S1P)/ceramide (Cer) ratio while 1,25(OH) 2 D 3 partially reverts this effect. 1,25(OH) 2 D 3 reverts also the Aβ(1−42)-induced reduction of sphingosine kinase activity. We have also studied the crosstalk between 1,25(OH) 2 D 3 and S1P signaling pathways downstream to the activation of S1P receptor subtype S1P1. Notably, we found that 1,25(OH) 2 D 3 prevents the reduction of S1P1 expression promoted by Aβ(1−42) and thereby it modulates the downstream signaling leading to ER stress damage (p38MAPK/ATF4). Similar effects were observed by using ZK191784. In addition, chronic treatment with 1,25(OH) 2 D 3 protects from aggregated Aβ(1−42)-induced damage in the CA1 region of the rat hippocampus and promotes cell proliferation in the hippocampal dentate gyrus of adult mice. In conclusion, these results represent the first evidence of the role of 1,25(OH) 2 D 3 and its structural analogue ZK191784 in counteracting the Aβ(1−42) peptide-induced toxicity through the modulation of S1P/S1P1/p38MAPK/ATF4 pathway in differentiated SH-SY5Y cells.

Tau is a microtubule-associated protein exerting several physiological functions in neurons. In Alzheimer's disease (AD) misfolded tau accumulates intraneuronally and leads to axonal degeneration. However, tau has also been found in the extracellular medium. Recent studies indicated that extracellular tau uploaded from neurons causes synaptic dysfunction and contributes to tau pathology propagation. Here we report novel evidence that extracellular tau oligomers are abundantly and rapidly accumulated in astrocytes where they disrupt intracellular Ca2+ signaling and Ca2+-dependent release of gliotransmitters, especially ATP. Consequently, synaptic vesicle release, the expression of pre- and post-synaptic proteins, and mEPSC frequency and amplitude were reduced in neighboring neurons. Notably, we found that tau uploading from astrocytes required the amyloid precursor protein, APP. Collectively, our findings suggests that astrocytes play a critical role in the synaptotoxic effects of tau via reduced gliotransmitter availability, and that astrocytes are major determinants of tau pathology in AD.

Down syndrome (DS) is the most diffused genetic cause of intellectual disability and, after the age of forty, is invariantly associated with Alzheimer's disease (AD). In the last years, the prolongation of life expectancy in people with DS renders the need for intervention paradigms aimed at improving mental disability and counteracting AD pathology particularly urgent. At present, however, there are no effective therapeutic strategies for DS and concomitant AD in mid-life people. The most intensively studied mouse model of DS is the Ts65Dn line, which summarizes the main hallmarks of the DS phenotype, included severe learning and memory deficits and age-dependent AD-like pathology. Here we report for the first time that middle-age Ts65Dn mice display a marked increase in soluble Aβ oligomer levels in their hippocampus. Moreover, we found that long-term exposure to environmental enrichment (EE), a widely used paradigm that increases sensory-motor stimulation, reduces Aβ oligomers and rescues spatial memory abilities in trisomic mice. Our findings underscore the potential of EE procedures as a non-invasive paradigm for counteracting brain aging processes in DS subjects.

Brain aging is characterized by global changes which are thought to underlie age-related cognitive decline. These include variations in brain activity and the progressive increase in the concentration of soluble amyloid-beta (A beta) oligomers, directly impairing synaptic function and plasticity even in the absence of any neurodegenerative disorder. Considering the high social impact of the decline in brain performance associated to aging, there is an urgent need to better understand how it can be prevented or contrasted. Lifestyle components, such as social interaction, motor exercise and cognitive activity, are thought to modulate brain physiology and its susceptibility to age-related pathologies. However, the precise functional and molecular factors that respond to environmental stimuli and might mediate their protective action again pathological aging still need to be clearly identified. To address this issue, we exploited environmental enrichment (EE), a reliable model for studying the effect of experience on the brain based on the enhancement of cognitive, social and motor experience, in aged wild-type mice. We analyzed the functional consequences of EE on aged brain physiology by performing in vivo local field potential (LFP) recordings with chronic implants. In addition, we also investigated changes induced by EE on molecular markers of neural plasticity and on the levels of soluble A beta oligomers. We report that EE induced profound changes in the activity of the primary visual and auditory cortices and in their functional interaction. At the molecular level, EE enhanced plasticity by an upward shift of the cortical excitation/inhibition balance. In addition, EE reduced brain A13 oligomers and increased synthesis of the A beta-degrading enzyme neprilysin. Our findings strengthen the potential of EE procedures as a non-invasive paradigm for counteracting brain aging processes.

Spinal cord injury (SCI) is a severe condition leading to enduring motor deficits. When lesions are incomplete, promoting spinal cord plasticity might be a useful strategy to elicit functional recovery. Here we investigated whether long-term fluoxetine administration in the drinking water, a treatment recently demonstrated to optimize brain plasticity in several pathological conditions, promotes motor recovery in rats that received a C4 dorsal funiculus crush. We show that fluoxetine administration markedly improved motor functions compared to controls in several behavioral paradigms. The improved functional effects correlated positively with significant sprouting of intact corticospinal fibers and a modulation of the excitation/inhibition balance. Our results suggest a potential application of fluoxetine treatment as a non invasive therapeutic strategy for SCI-associated neuropathologies.

SummaryThe positioning of neurons in the cerebral cortex is of crucial importance for its function as highlighted by the severe consequences of migrational disorders in patients. Here we show that genetic deletion of the small GTPase RhoA in the developing cerebral cortex results in two migrational disorders: subcortical band heterotopia (SBH), a heterotopic cortex underlying the normotopic cortex, and cobblestone lissencephaly, in which neurons protrude beyond layer I at the pial surface of the brain. Surprisingly, RhoA−/− neurons migrated normally when transplanted into wild-type cerebral cortex, whereas the converse was not the case. Alterations in the radial glia scaffold are demonstrated to cause these migrational defects through destabilization of both the actin and the microtubules cytoskeleton. These data not only demonstrate that RhoA is largely dispensable for migration in neurons but also showed that defects in radial glial cells, rather than neurons, can be sufficient to produce SBH.

Environmental enrichment (EE) is an experimental protocol based on a complex sensorimotor stimulation that dramatically affects brain development. While it is widely believed that the effects of EE result from the unique combination of different sensory and motor stimuli, it is not known whether and how cortico-cortical interactions are shaped by EE. Since the primary visual cortex (V1) is one of the best characterized targets of EE, we looked for direct cortico-cortical projections impinging on V1, and we identified a direct monosynaptic connection between motor cortex and V1 in the mouse brain. To measure the interactions between these areas under standard and EE rearing conditions, we used simultaneous recordings of local field potentials (LFPs) in awake, freely moving animals. LFP signals were analyzed by using different methods of linear and nonlinear analysis of time series (cross-correlation, mutual information, phase synchronization). We found that EE decreases the level of coupling between the electrical activities of the two cortical regions with respect to the control group. From a functional point of view, our results indicate, for the first time, that an enhanced sensorimotor experience impacts on the brain by affecting the functional crosstalk between different cortical areas.

It has been demonstrated that the complex sensorimotor and social stimulation achieved by rearing animals in an enriched environment (EE) can reinstate juvenile-like plasticity in the adult cortex. However, it is not known whether EE can affect thalamocortical transmission. Here, we recorded in vivo field potentials from the visual cortex evoked by electrical stimulation of the dorsal lateral geniculate nucleus (dLGN) in anesthetized rats. We found that a period of EE during adulthood shifted the input-output curves and increased paired-pulse depression, suggesting an enhanced synaptic strength at thalamocortical terminals. Accordingly, EE animals showed an increased expression of the vesicular glutamate transporter 2 (vGluT-2) in geniculocortical afferents to layer IV. Rats reared in EE also showed an enhancement of thalamocortical long-term potentiation (LTP) triggered by theta-burst stimulation (TBS) of the dLGN. To monitor the functional consequences of increased LTP in EE rats, we recorded visual evoked potentials (VEPs) before and after application of TBS to the geniculocortical pathway. We found that responses to visual stimulation were enhanced across a range of contrasts in EE animals. This was accompanied by an up-regulation of the intracortical excitatory synaptic marker vGluT-1 and a decrease in the expression of the vesicular GABA transporter (vGAT), indicating a shift in the excitation/inhibition ratio. Thus, in the adult rat, EE enhances synaptic strength and plasticity of the thalamocortical pathway associated with specific changes in glutamatergic and GABAergic neurotransmission. These data provide novel insights into the mechanisms by which EE shapes the adult brain. (c) 2010 Wiley-Liss, Inc.

Western lifestyle contributes to body weight dysregulation. Leptin down-regulates food intake by modulating the activity of neural circuits in the hypothalamic arcuate nucleus (ARC), and resistance to this hormone constitutes a permissive condition for obesity. Physical exercise modulates leptin sensitivity in diet-induced obese rats. The role of other lifestyle components in modulating leptin sensitivity remains elusive. Environmentally enriched mice were used to explore the effects of lifestyle change on leptin production/action and other metabolic parameters. We analyzed adult mice exposed to environmental enrichment (EE), which showed decreased leptin, reduced adipose mass, and increased food intake. We also analyzed 50-d-old mice exposed to either EE (YEE) or physical exercise (YW) since birth, both of which showed decreased leptin. YEE mice showed no change in food intake, increased response to leptin administration, increased activation of STAT3 in the ARC. The YW leptin-induced food intake response was intermediate between young mice kept in standard conditions and YEE. YEE exhibited increased and decreased ratios of excitatory/inhibitory synapses onto α-melanocyte-stimulating hormone and agouti-related peptide neurons of the ARC, respectively. We also analyzed animals as described for YEE and then placed in standard cages for 1 mo. They showed no altered leptin production/action but demonstrated changes in excitatory/inhibitory synaptic contacts in the ARC similar to YEE. EE and physical activity resulted in improved insulin sensitivity. In conclusion, EE and physical activity had an impact on feeding behavior, leptin production/action, and insulin sensitivity, and EE affected ARC circuitry. The leptin-hypothalamic axis is maximally enhanced if environmental stimulation is applied during development.

Background. Neurorehabilitation protocols based on the use of robotic devices have recently shown to provide promising clinical results. However, their efficacy is still limited because of the poor comprehension of the mechanisms at the basis of functional enhancements. Objective. To increase basic understanding of robot-mediated neurorehabilitation by performing experiments on a rodent model of stroke. Methods. Mice were trained to pull back a handle on a robotic platform and their performances in the task were evaluated before and after a focal cortical ischemic stroke. The platform was designed for the quantitative assessment of forelimb function via a series of parameters (time needed to complete the task, t-target; average force; number of sub-movements). Results. The animals rapidly learned the retraction task and reached asymptotic performance by the fifth session of training. Within 2 to 6 days after a small, endothelin-1-induced lesion in the caudal forelimb area, mice showed an increase in t-target and number of sub-movements and a corresponding decrease in the average force exerted. These parameters returned to baseline, pre-lesion values with continued platform training (10-14 days after stroke). Conclusions. These results highlight the utility of the devised platform for characterizing post-infarct deficits and improvements of forelimb performance. Further research is warranted to widen the understanding of device-dependent rehabilitation effects.