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This presentation has been given at the 3rd General Meeting of the project ECOPOTENTIAL (Matalasca��as, Spain, 2018) and it shows the progress made and the results of the assessment of the threats that affect the habitat of the iconic species Salmo letnica.

Objectives GDI1 gene encodes for αGDI, a protein controlling the cycling of small GTPases, reputed to orchestrate vesicle trafficking. Mutations in human GDI1 are responsible for intellectual disability (ID). In mice with ablated Gdi1, a model of ID, impaired working and associative short-term memory was recorded. This cognitive phenotype worsens if the deletion of αGDI expression is restricted to neurons. However, whether astrocytes, key homeostasis providing neuroglial cells, supporting neurons via aerobic glycolysis, contribute to this cognitive impairment is unclear. Methods We carried out proteomic analysis and monitored [18F]-fluoro-2-deoxy-d-glucose uptake into brain slices of Gdi1 knockout and wild type control mice. d-Glucose utilization at single astrocyte level was measured by the Förster Resonance Energy Transfer (FRET)-based measurements of cytosolic cyclic AMP, d-glucose and L-lactate, evoked by agonists selective for noradrenaline and L-lactate receptors. To test the role of astrocyte-resident processes in disease phenotype, we generated an inducible Gdi1 knockout mouse carrying the Gdi1 deletion only in adult astrocytes and conducted behavioural tests. Results Proteomic analysis revealed significant changes in astrocyte-resident glycolytic enzymes. Imaging [18F]-fluoro-2-deoxy-d-glucose revealed an increased d-glucose uptake in Gdi1 knockout tissue versus wild type control mice, consistent with the facilitated d-glucose uptake determined by FRET measurements. In mice with Gdi1 deletion restricted to astrocytes, a selective and significant impairment in working memory was recorded, which was rescued by inhibiting glycolysis by 2-deoxy-d-glucose injection. Conclusions These results reveal a new astrocyte-based mechanism in neurodevelopmental disorders and open a novel therapeutic opportunity of targeting aerobic glycolysis, advocating a change in clinical practice., Highlights • Mutations in human Gdi1, encoding αGDI, a protein controlling vesicle traffic, are responsible for Intellectual Disability. • Gdi1 knockout revealed significant changes in astrocyte-resident glycolytic enzymes and facilitated D-glucose utilization. • Astrocyte-selective Gdi1 deletion impairs working memory, which can be rescued by administration of 2-deoxy-D-glucose. • Astrocyte-based glycolysis is a new target to treat Intellectual Disability.

The development of a tractable small animal model faithfully reproducing human coronavirus disease 2019 pathogenesis would arguably meet a pressing need in biomedical research. Thus far, most investigators have used transgenic mice expressing the human ACE2 in epithelial cells (K18-hACE2 transgenic mice) that are intranasally instilled with a liquid severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) suspension under deep anesthesia. Unfortunately, this experimental approach results in disproportionate high central nervous system infection leading to fatal encephalitis, which is rarely observed in humans and severely limits this model’s usefulness. Here, we describe the use of an inhalation tower system that allows exposure of unanesthetized mice to aerosolized virus under controlled conditions. Aerosol exposure of K18-hACE2 transgenic mice to SARS-CoV-2 resulted in robust viral replication in the respiratory tract, anosmia, and airway obstruction but did not lead to fatal viral neuroinvasion. When compared with intranasal inoculation, aerosol infection resulted in a more pronounced lung pathology including increased immune infiltration, fibrin deposition, and a transcriptional signature comparable to that observed in SARS-CoV-2–infected patients. This model may prove useful for studies of viral transmission, disease pathogenesis (including long-term consequences of SARS-CoV-2 infection), and therapeutic interventions.

Spinal cord injury (SCI) is a devastating condition mainly deriving from a traumatic damage of the spinal cord (SC). Immune cells and endogenous SC-neural stem cells (SC-NSCs) play a critical role in wound healing processes, although both are ineffective to completely restore tissue functioning. The role of SC-NSCs in SCI and, in particular, whether such cells can interplay with the immune response are poorly investigated issues, although mechanisms governing such interactions might open new avenues to develop novel therapeutic approaches. Background: Spinal cord injury (SCI) is a devastating condition mainly deriving from a traumatic damage of the spinal cord (SC). Immune cells and endogenous SC-neural stem cells (SC-NSCs) play a critical role in wound healing processes, although both are ineffective to completely restore tissue functioning. The role of SC-NSCs in SCI and, in particular, whether such cells can interplay with the immune response are poorly investigated issues, although mechanisms governing such interactions might open new avenues to develop novel therapeutic approaches. Methods: We used two transgenic mouse lines to trace as well as to kill SC-NSCs in mice receiving SCI. We used Nestin CreERT2 mice to trace SC-NSCs descendants in the spinal cord of mice subjected to SCI. While mice carrying the suicide gene thymidine kinase (TK) along with the GFP reporter, under the control of the Nestin promoter regions (NestinTKmice) were used to label and selectively kill SC-NSCs. Results: We found that SC-NSCs are capable to self-activate after SCI. In addition, a significant worsening of clinical and pathological features of SCI was observed in the NestinTKmice, upon selective ablation of SC-NSCs before the injury induction. Finally, mice lacking in SC-NSCs and receiving SCI displayed reduced levels of different neurotrophic factors in the SC and significantly higher number of M1-like myeloid cells. Conclusion: Our data show that SC-NSCs undergo cell proliferation in response to traumatic spinal cord injury. Mice lacking SC-NSCs display overt microglia activation and exaggerate expression of pro-inflammatory cytokines. The absence of SC-NSCs impaired functional recovery as well as neuronal and oligodendrocyte cell survival. Collectively our data indicate that SC-NSCs can interact with microglia/macrophages modulating their activation/responses and that such interaction is importantly involved in mechanisms leading tissue recovery.

Adenosine Deaminase (ADA) deficiency is an autosomal recessive variant of severe combined immunodeficiency (SCID) caused by systemic accumulation of ADA substrates. Neurological and behavioral abnormalities observed in ADA-SCID patients surviving after stem cell transplantation or gene therapy represent an unresolved enigma in the field. We found significant neurological and cognitive alterations in untreated ADA-SCID patients as well as in two groups of patients after short- and long-term enzyme replacement therapy with PEG-ADA. These included motor dysfunction, EEG alterations, sensorineural hypoacusia, white matter and ventricular alterations in MRI as well as a low mental development index or IQ. Ada-deficient mice were significantly less active and showed anxiety-like behavior. Molecular and metabolic analyses showed that this phenotype coincides with metabolic alterations and aberrant adenosine receptor signaling. PEG-ADA treatment corrected metabolic adenosine-based alterations, but not cellular and signaling defects, indicating an intrinsic nature of the neurological and behavioral phenotype in ADA deficiency.

Background RAB39B pathogenic variants cause X-linked Parkinsonism associated with Intellectual Disability, known as Waisman syndrome, a very rare disorder that has been mainly identified through exome sequencing in large Parkinson's disease cohorts. In this study we searched for pathogenic variants in RAB39B in two Italian families affected by X-linked early-onset Parkinsonism and Intellectual Disability. Methods Three patients received neurological evaluation and underwent RAB39B sequencing. Results Two novel RAB39B frameshift variants were found to result in the absence of RAB39B protein (family 1: c.137dupT; family 2: c.371delA). Patients showed unilateral rest tremor and bradykinesia; one of them also displayed an early-onset postural tremor. Paramagnetic substance deposition in the substantia nigra, globus pallidi, red nucleus, putamen and pulvinar was assessed by brain imaging. Two patients also showed moderate calcification of globus pallidi. Conclusion In this study we highlight the evidence that X-linked early-onset Parkinsonism associated with Intellectual Disability occurs as a pattern of clinical and neuroimaging features attributable to RAB39B pathogenic variants.

The search for biomarkers of antidepressant effects focused on pathways regulating synaptic plasticity, and on activated inflammatory markers. Repeated Sleep Deprivation (SD) provides a model treatment to reverse-translate antidepressant effects from in vivo clinical psychiatry to model organisms. We studied the effects of repeated SD alone (ASD) or combined with exercise on a slow spinning wheel (SSW), in 116 C57BL/6J male mice divided in three groups (ASD, SSW, untreated). Forced Swimming Test (FST) was used to detect antidepressant-like effects. Unbiased evaluation of the transcriptional responses were obtained in the hippocampus by Illumina Bead Chip Array system, then confirmed with real time PCR. Spine densities in granular neurons of the dentate gyrus (DG) were assayed by standard Golgi staining. Activation of Microglial/Macrophages cells was evaluated by immunufluorescence analysis for Iba1. Rates of cell proliferation was estimated pulsing mice with the S-phase tracer 5-Iodo-2'-deoxyuridine (IdU). All SD procedures caused a decreasing of floating time at FST, and increased expression of the immediate early gene Arc/Arg3.1. In addition, SSW also increased expression of the Microglia/Macrophages genes Iba-1 and chemokine receptors Cx3cR1 and CxcR4, of the canonical Wnt signaling gene Wnt7a, and of dendritic spines in CA4 neurons of the DG. SSW up-regulated both the number of Iba1+ cells and rates of cell proliferation in the subgranular region of the DG. The antidepressant-like effects of SD dissociated both, from hippocampal neuroplasticity in the DG (not occurring after ASD), and from microglial activation (not preventing behavioral response when occurring). The increase in dendritic spine density in the DG after SD and exercise was associated with an up-regulation of Wnt 7a, and with activation of the innate immune system of the brain. Increased Arc/Arg3.1 suggests however increased neuroplasticity, which could be common to all fast-acting antidepressants, and possibly occurring in other brain areas.

Contains fulltext : 126190.pdf (Publisher’s version ) (Open Access) We recently reported that duplication of the E3 ubiquitin ligase HUWE1 results in intellectual disability (ID) in male patients. However, the underlying molecular mechanism remains unknown. We used Drosophila melanogaster as a model to investigate the effect of increased HUWE1 levels on the developing nervous system. Similar to the observed levels in patients we overexpressed the HUWE1 mRNA about 2-fold in the fly. The development of the mushroom body and neuromuscular junctions were not altered, and basal neurotransmission was unaffected. These data are in agreement with normal learning and memory in the courtship conditioning paradigm. However, a disturbed branching phenotype at the axon terminals of the dorsal cluster neurons (DCN) was detected. Interestingly, overexpression of HUWE1 was found to decrease the protein levels of dishevelled (dsh) by 50%. As dsh as well as Fz2 mutant flies showed the same disturbed DCN branching phenotype, and the constitutive active homolog of beta-catenin, armadillo, could partially rescue this phenotype, our data strongly suggest that increased dosage of HUWE1 compromises the Wnt/beta-catenin pathway possibly by enhancing the degradation of dsh.

The functional significance of adult neural stem and progenitor cells in hippocampal-dependent learning and memory has been well documented. Although adult neural stem and progenitor cells in the subventricular zone are known to migrate to, maintain and reorganize the olfactory bulb, it is less clear whether they are functionally required for other processes. Using a conditional transgenic mouse model, selective ablation of adult neural stem and progenitor cells in the subventricular zone induced a dramatic increase in morbidity and mortality of central nervous system disorders characterized by excitotoxicity-induced cell death accompanied by reactive inflammation, such as 4-aminopyridine-induced epilepsy and ischaemic stroke. To test the role of subventricular zone adult neural stem and progenitor cells in protecting central nervous system tissue from glutamatergic excitotoxicity, neurophysiological recordings of spontaneous excitatory postsynaptic currents from single medium spiny striatal neurons were measured on acute brain slices. Indeed, lipopolysaccharide-stimulated, but not unstimulated, subventricular zone adult neural stem and progenitor cells reverted the increased frequency and duration of spontaneous excitatory postsynaptic currents by secreting the endocannabinod arachidonoyl ethanolamide, a molecule that regulates glutamatergic tone through type 1 cannabinoid receptor (CB(1)) binding. In vivo restoration of cannabinoid levels, either by administration of the type 1 cannabinoid receptor agonist HU210 or the inhibitor of the principal catabolic enzyme fatty acid amide hydrolase, URB597, completely reverted the increased morbidity and mortality of adult neural stem and progenitor cell-ablated mice suffering from epilepsy and ischaemic stroke. Our results provide the first evidence that adult neural stem and progenitor cells located within the subventricular zone exert an 'innate' homeostatic regulatory role by protecting striatal neurons from glutamate-mediated excitotoxicity.

"The GDI1 gene encodes αGDI, which retrieves inactive GDP-bound RAB from membranes to form a cytosolic pool awaiting vesicular release. Mutations in GDI1 are responsible for X-linked Intellectual Disability. Characterization of the Gdi1-null mice has revealed alterations in the total number and distribution of hippocampal and cortical synaptic vesicles, hippocampal short-term synaptic plasticity and specific short-term memory deficits in adult mice, which are possibly caused by alterations of different synaptic vesicle recycling pathways controlled by several RAB GTPases. However, interpretation of these studies is complicated by the complete ablation of Gdi1 in all cells in the brain throughout development. In this study, we generated conditionally gene-targeted mice in which the knockout of Gdi1 is restricted to the forebrain, hippocampus, cortex and amygdala and occurs only during postnatal development. Adult mutant mice reproduce the short-term memory deficit previously reported in Gdi1-null mice. Surprisingly, the delayed ablation of Gdi1 worsens the pre-synaptic phenotype at cortico-amygdala synaptic connections compared to Gdi1-null mice. These results suggest a pivotal role of αGDI via specific RAB GTPases acting specifically in forebrain regions at the pre-synaptic sites involved in memory formation."

Protein synthesis is a tightly regulated, energy-consuming process. The control of mRNA translation into protein is fundamentally important for the fine-tuning of gene expression; additionally, precise translational control plays a critical role in many cellular processes, including development, cellular growth, proliferation, differentiation, synaptic plasticity, memory, and learning. Eukaryotic translation initiation factor 4h ( Eif4h ) encodes a protein involved in the process of protein synthesis, at the level of initiation phase. Its human homolog, WBSCR1 , maps on 7q11.23, inside the 1.6 Mb region that is commonly deleted in patients affected by the Williams-Beuren syndrome, which is a complex neurodevelopmental disorder characterized by cardiovascular defects, cerebral dysplasias and a peculiar cognitive-behavioral profile. In this study, we generated knockout mice deficient in Eif4h . These mice displayed growth retardation with a significant reduction of body weight that began from the first week of postnatal development. Neuroanatomical profiling results generated by magnetic resonance imaging analysis revealed a smaller brain volume in null mice compared with controls as well as altered brain morphology, where anterior and posterior brain regions were differentially affected. The inactivation of Eif4h also led to a reduction in both the number and complexity of neurons. Behavioral studies revealed severe impairments of fear-related associative learning and memory formation. These alterations suggest that Eif4h might contribute to certain deficits associated with Williams-Beuren syndrome.