One of the main challenges in cancer treatment is addressing the metabolic reprogramming of tumor cells, which require more energy and biomolecules than healthy cells. Cancer cells alter their metabolism by switching between glycolysis and oxidative phosphorylation (OXPHOS). These processes depend on transmembrane proteins that respond to the extracellular environment. Our research identified the transmembrane form of Chloride Intracellular Channel 1 (tmCLIC1) as a marker of malignancy and a potential therapeutic target. tmCLIC1 levels are increased in several solid tumors, supporting cancer growth and progression, whereas they are mostly absent in healthy cells. We confirmed that tmCLIC1 is the specific target of the antidiabetic drug metformin, an OXPHOS inhibitor in cancer cells. tmCLIC1 is the primary target of metformin in glioblastoma-initiating cells, as shown by single-channel patch-clamp recordings and NMR experiments. Various patient-derived glioblastoma cells with different genetic backgrounds were used to demonstrate that CLIC1 CRISPR-Cas9 knockout and/or its point mutation at arginine 29 removes metformin’s antitumor effects. Functional assays were used to assess the effects on proliferation, mitochondrial metabolism, and tumor growth in vitro and in vivo, using zebrafish and murine xenograft models. Metformin inhibits the function of tmCLIC1 through direct and specific binding involving arginine 29 in the tmCLIC1 sequence. Additionally, during hypoglycemia, metformin promotes glioblastoma cell apoptosis by inhibiting the Cancerous Inhibitor of Protein Phosphatase 2 A (CIP2A) and activating the PP2A B56δ subunit. This leads to the dephosphorylation of Glycogen Synthase Kinase 3 Beta (GSK3β), resulting in the breakdown of the pro-survival protein MCL-1 and subsequent cell death. Inhibition of tmCLIC1 is crucial for this metformin-driven antineoplastic effect, mainly through regulating the PP2A-GSK3β-MCL-1 pathway under hypoglycemic conditions. The chronic presence of metformin within the tumors impairs in vivo growth at nanomolar concentrations. The therapeutic role of metformin to treat brain tumors remains debated. Our findings show that drug delivery is essential, as in vivo, tumor growth decreases at concentrations below 10 nanomolar. We propose that sustained CNS metformin levels may improve tmCLIC1 inhibition, providing a basis for optimizing interactions with metformin or related compounds to enhance therapeutic efficacy.

The serotonergic system originates from a small population of brainstem neurons whose axons form one of the most extensive projection networks in the vertebrate nervous system. Although a number of molecular regulators of serotonergic development have been identified, the core principles that organize this widespread axonal architecture remain poorly understood. Classical neuroanatomical studies have proposed that serotonergic axons may navigate by growing along pre-existing fiber tracts, a process termed epiphytic guidance, but this hypothesis has remained largely untested. Here, we used organotypic transplantation assays to investigate how embryonic serotonergic axons navigate within developing and adult neural tissue. Rostral raphe explants from Tph2-GFP embryos were grafted onto embryonic hindbrain flat-mounts or adult brain slices, allowing donor-derived axons to be visualized in relation to genetically labelled endogenous serotonergic pathways and host tissue architecture. Across different grafting configurations, developmental stages, and both homotopic and heterotopic host territories, donor-derived axons consistently aligned with pre-existing serotonergic pathways adopting local trajectories rather than growing independently of them. This substrate-dependent behavior extended to adult tissue, where transplanted embryonic axons preferentially followed pre-existing serotonergic axon directions. These observations reveal substantial navigational plasticity and indicate that axonal and tissue architectures can provide permissive and potentially instructive substrates for the dispersal of serotonergic fibers. Together, our findings provide experimental support for epiphytic guidance and suggest that serotonergic pathway assembly relies, at least in part, on a pioneer-follower mechanism of progressive self-scaffolding. Such a strategy may help explain how a small population of raphe neurons generates an extensive and spatially coherent neuromodulatory system.

The prefrontal cortex (PFC) and locus coeruleus (LC) form a bidirectional circuit essential for cognitive control and arousal. LC-derived norepinephrine (NE) influences PFC network dynamics, yet the mechanisms by which descending PFC inputs regulate LC activity and gate its neuromodulatory feedback remain unclear. Here, we describe an anatomical and functional PFC-LC loop subject to endocannabinoid (eCB) negative feedback. Optogenetic activation of PFC terminals in the LC enhances NE release and recruits NE-sensitive neuronal activity in the PFC. Under behaviorally relevant patterns of activation, LC-derived eCBs are mobilized, thereby weakening PFC-to-LC synaptic transmission. This limits subsequent NE release in the PFC and constrains the recruitment of NE-sensitive PFC neuronal assemblies. Modulation of this PFC-LC interaction shapes outcome discrimination in place conditioning tasks. By elucidating how eCB signaling modulates the PFC-LC loop, our findings reveal how these lipid molecules fine-tune NE signaling in the PFC, which is involved in cognitive and arousal-related processes.

Chromosome 22q11.2 deletion increases the risk of neuropsychiatric disorders like autism and schizophrenia. Disruption of large-scale functional connectivity in 22q11 deletion syndrome (22q11DS) has been widely reported, but the biological factors driving these changes remain unclear. We used a cross-species design to uncover the developmental trajectory and neural underpinnings of brain dysconnectivity in 22q11DS. In LgDel mice, a model for 22q11DS, we found age-specific patterns of brain dysconnectivity, with widespread fMRI hyperconnectivity in juvenile mice reconfiguring to hippocampal hypoconnectivity over puberty. These changes correlated with developmental alterations in dendritic spine density, and both were transiently normalized by GSK3β inhibition, suggesting a synaptic origin for this phenomenon. Notably, analogous pubertal hyperconnectivity-to-hypoconnectivity reconfiguration occurs in human 22q11DS, affecting cortical regions enriched for GSK3β-associated synaptic genes and autism-relevant transcripts. This dysconnectivity also predicts age-dependent social alterations in 22q11DS individuals. These results suggest that synaptic mechanisms underlie developmental brain dysconnectivity in 22q11DS.

Chromosome 22q11.2 deletion is among the strongest known genetic risk factors for neuropsychiatric disorders, including autism and schizophrenia. Brain imaging studies have reported disrupted large-scale functional connectivity in people with 22q11 deletion syndrome (22q11DS). However, the significance and biological determinants of these functional alterations remain unclear. Here, we use a cross-species design to investigate the developmental trajectory and neural underpinnings of brain dysconnectivity in 22q11DS. We find that LgDel mice, an established mouse model of 22q11DS, exhibit age-specific patterns of functional MRI (fMRI) dysconnectivity, with widespread fMRI hyper-connectivity in juvenile mice reverting to focal hippocampal hypoconnectivity over puberty. These fMRI connectivity alterations are mirrored by co-occurring developmental alterations in dendritic spine density, and are both transiently normalized by developmental GSK3β inhibition, suggesting a synaptic origin for this phenomenon. Notably, analogous hyper-to hypoconnectivity reconfiguration occurs also in human 22q11DS, where it affects hippocampal and cortical regions spatially enriched for synaptic genes that interact with GSK3β, and autism-relevant transcripts. Functional dysconnectivity in somatomotor components of this network is predictive of age-dependent social alterations in 22q11.2 deletion carriers. Taken together, these findings suggest that synaptic-related mechanisms underlie developmentally mediated functional dysconnectivity in 22q11DS.

Abstract BACKGROUND The interaction between tumor cells and the main components of the tumor microenvironment (TME) seems to guide the progression of glioma. Several recent studies have highlighted that the synaptic activity of pyramidal neurons potently drives Glioblastoma (GB) proliferation. However, the role played by other neuronal cell types is still poorly understood. Based on our previous findings showing that the optogenetic stimulation of parvalbuminergic (PV)-inhibitory interneurons negatively regulates glioma progression, here we tested whether a chemogenetic manipulation of peritumoral PV-interneurons could influence GB growth. MATERIAL AND METHODS To address this issue, AAV-hSyn-DIO-hM4D(Gi)-mCherry or AAV-hSyn-DIO-hM4D(Gq)-mCherry were injected in the motor cortex bilaterally with respect to the GL261 injection site in PV-Cre mice. In order to manipulate PV-interneurons activity, animals will be randomized to receive CNO or vehicle intraperitoneally twice a day from day 12 (i.e., the glioma symptomatic stage) to day 22 (i.e., the glioma late stage) post tumor induction. As readouts, we: i) evaluated the tumor volume using Magnetic Resonance Imaging (MRI), ii) performed a longitudinal monitoring of motor capabilities using specific motor tests (i.e., Grip Strength and Grid walk), iii) assessed the survival, iv) determined the GB proliferation index of glioma-bearing mice after different treatments. RESULTS We found that the PV-interneurons inactivation resulted in worsening motor performances with respect to the vehicle mice. Despite that, no difference was observed in animal median survival or tumor volume among the two groups. However, MRI data showed a very significative reduction in the tumor volume at 28 days after glioma induction in PV-activated glioma bearing mice (p=0,004). In addition, PV-activation exert a slight effect on the maximum survival of CNO-treated glioma bearing mice with respect to the vehicle mice (39 vs 32 days). CONCLUSION Since there is no cure for GB, it is essential to find new and effective therapeutic approaches to improve the quality of life and to extend the life expectancy in patients. These data suggest that the inhibitory circuitry in the tumor-adjacent zone could delay GB growth, protecting peritumoral tissue from GB proliferation.

Cre-lox system is a major tool in mouse molecular genetics instrumental in promoting somatic recombination to spatiotemporally control transcriptional activation/inhibition in subsets of cells or tissues in vivo. A critical factor behind this system may be represented by the availability of a specific promoter driving Cre expression in the cell subset of interest. Split-Cre recombinase system represents an evolution that circumvents this limitation using split N- and C-terminal domains of Cre recombinase placed under the control of two distinct promoters defining an intersectional domain where functional complementation of Cre protein fragments is obtained. This system is a valuable tool for controlling Cre recombinase activity in a spatially and temporally defined manner based on the assumption that neither N- or C-terminal Cre fragments alone have recombinase activity. However, residual recombinase activity of one of the two fragments can occur leading to confounding experimental results. In this work, we delve into characterizing functional activity of different N-terminal deleted codon-optimized Cre (iCre) isoforms to refine Split-Cre-based technologies, aiming to avoid uncontrolled recombinase events. Given the presence of several methionine residues in the amino acidic iCre sequence, we explored whether these residues could serve as potential translation start sites, resulting in truncated isoforms that might retain recombinase activity. To address this question, we tested in HEK293T cells whether site-specific recombination was retained in progressively amino-terminal deleted iCre isoforms. Our results reveal residual enzymatic activity of most amino-terminal deleted isoforms of iCre whose ATG start codon is located downstream of the commonly used split site. This insight holds significance for future refinements of the widely used Split-Cre system, providing information to avoid false positive results stemming from unwanted activity.

Serotonin-releasing fibers depart from the raphe nuclei to profusely innervate the entire central nervous system, displaying in some brain regions high structural plasticity in response to genetically induced abrogation of serotonin synthesis. Chronic fluoxetine treatment used as a tool to model peri-physiological, clinically relevant serotonin elevation is also able to cause structural rearrangements of the serotonergic fibers innervating the hippocampus. Whether this effect is limited to hippocampal-innervating fibers or extends to other populations of axons is not known. Here, we used confocal imaging and three-dimensional (3-D) modeling analysis to expand our morphological investigation of fluoxetine-mediated effects on serotonergic circuitry. We found that chronic treatment with a behaviorally active dose of fluoxetine affects the morphology and reduces the density of serotonergic axons innervating the medial prefrontal cortex, a brain region strongly implicated in the regulation of depressive- and anxiety-like behavior. Axons innervating the somatosensory cortex were unaffected, suggesting differential susceptibility to serotonin changes across cortical areas. Importantly, a 1-month washout period was sufficient to reverse morphological changes in both the medial prefrontal cortex and in the previously characterized hippocampus, as well as to normalize behavior, highlighting an intriguing relationship between axon density and an antidepressant-like effect. Overall, these results further demonstrate the bidirectional plasticity of defined serotonergic axons and provide additional insights into fluoxetine effects on the serotonergic system.

Physiology and behavior are structured temporally to anticipate daily cycles of light and dark, ensuring fitness and survival. Neuromodulatory systems in the brain—including those involving serotonin and dopamine—exhibit daily oscillations in neural activity and help shape circadian rhythms. Disrupted neuromodulation can cause circadian abnormalities that are thought to underlie several neuropsychiatric disorders, including bipolar mania and schizophrenia, for which a mechanistic understanding is still lacking. Here, we show that genetically depleting serotonin in Tph2 knockout mice promotes manic-like behaviors and disrupts daily oscillations of the dopamine biosynthetic enzyme tyrosine hydroxylase (TH) in midbrain dopaminergic nuclei. Specifically, while TH mRNA and protein levels in the Substantia Nigra (SN) and Ventral Tegmental Area (VTA) of wild-type mice doubled between the light and dark phase, TH levels were high throughout the day in Tph2 knockout mice, suggesting a hyperdopaminergic state. Analysis of TH expression in striatal terminal fields also showed blunted rhythms. Additionally, we found low abundance and blunted rhythmicity of the neuropeptide cholecystokinin (Cck) in the VTA of knockout mice, a neuropeptide whose downregulation has been implicated in manic-like states in both rodents and humans. Altogether, our results point to a previously unappreciated serotonergic control of circadian dopamine signaling and propose serotonergic dysfunction as an upstream mechanism underlying dopaminergic deregulation and ultimately maladaptive behaviors.

Genomic mechanisms enhancing risk in males may contribute to sex bias in autism. The ubiquitin protein ligase E3A gene ( Ube3a ) affects cellular homeostasis via control of protein turnover and by acting as transcriptional coactivator with steroid hormone receptors. Overdosage of Ube3a via duplication or triplication of chromosomal region 15q11-13 causes 1 to 2% of autistic cases. Here, we test the hypothesis that increased dosage of Ube3a may influence autism-relevant phenotypes in a sex-biased manner. We show that mice with extra copies of Ube3a exhibit sex-biasing effects on brain connectomics and autism-relevant behaviors. These effects are associated with transcriptional dysregulation of autism-associated genes, as well as genes differentially expressed in 15q duplication and in autistic people. Increased Ube3a dosage also affects expression of genes on the X chromosome, genes influenced by sex steroid hormone, and genes sex-differentially regulated by transcription factors. These results suggest that Ube3a overdosage can contribute to sex bias in neurodevelopmental conditions via influence on sex-differential mechanisms.

The D-aspartate oxidase (DDO) gene encodes the enzyme responsible for the catabolism of D-aspartate, an atypical amino acid enriched in the mammalian brain and acting as an endogenous NMDA receptor agonist. Considering the key role of NMDA receptors in neurodevelopmental disorders, recent findings suggest a link between D-aspartate dysmetabolism and schizophrenia. To clarify the role of D-aspartate on brain development and functioning, we used a mouse model with constitutive Ddo overexpression and D-aspartate depletion. In these mice, we found reduced number of BrdU-positive dorsal pallium neurons during corticogenesis, and decreased cortical and striatal gray matter volume at adulthood. Brain abnormalities were associated with social recognition memory deficit at juvenile phase, suggesting that early D-aspartate occurrence influences neurodevelopmental related phenotypes. We corroborated this hypothesis by reporting the first clinical case of a young patient with severe intellectual disability, thought disorders and autism spectrum disorder symptomatology, harboring a duplication of a chromosome 6 region, including the entire DDO gene.

Postmortem studies have revealed increased density of excitatory synapses in the brains of individuals with autism spectrum disorder (ASD), with a putative link to aberrant mTOR-dependent synaptic pruning. ASD is also characterized by atypical macroscale functional connectivity as measured with resting-state fMRI (rsfMRI). These observations raise the question of whether excess of synapses causes aberrant functional connectivity in ASD. Using rsfMRI, electrophysiology and in silico modelling in Tsc2 haploinsufficient mice, we show that mTOR-dependent increased spine density is associated with ASD -like stereotypies and cortico-striatal hyperconnectivity. These deficits are completely rescued by pharmacological inhibition of mTOR. Notably, we further demonstrate that children with idiopathic ASD exhibit analogous cortical-striatal hyperconnectivity, and document that this connectivity fingerprint is enriched for ASD-dysregulated genes interacting with mTOR or Tsc2. Finally, we show that the identified transcriptomic signature is predominantly expressed in a subset of children with autism, thereby defining a segregable autism subtype. Our findings causally link mTOR-related synaptic pathology to large-scale network aberrations, revealing a unifying multi-scale framework that mechanistically reconciles developmental synaptopathy and functional hyperconnectivity in autism.

Introduction: Plasticity at corticostriatal synapses is a key substrate for a variety of brain functions - including motor control, learning and reward processing - and is often disrupted in disease conditions. Despite intense research pointing toward a dynamic interplay between glutamate, dopamine (DA), and serotonin (5-HT) neurotransmission, their precise circuit and synaptic mechanisms regulating their role in striatal plasticity are still unclear. Here, we analyze the role of serotonergic raphe-striatal innervation in the regulation of DA-dependent corticostriatal plasticity. Methods: Mice (males and females, 2-6 months of age) were housed in standard plexiglass cages at constant temperature (22 +/- 1 degrees C) and maintained on a 12/12 h light/dark cycle with food and demineralized water ad libitum. In the present study, we used a knock-in mouse line in which the green fluorescent protein reporter gene (GFP) replaced the I Tph2 exon (Tph2(GFP) mice), allowing selective expression of GFP in the whole 5-HT system, highlighting both somata and neuritis of serotonergic neurons. Heterozygous, Tph2(+/)(GFP), mice were intercrossed to obtain experimental cohorts, which included Wild-type (Tph2(+/+)), Heterozygous (Tph2(+/)(GFP)), and Mutant serotonin-depleted (Tph2(GFP)(/)(GFP)) animals. Results: Using male and female mice, carrying on different Tph2 gene dosages, we show that Tph2 gene modulation results in sex-specific corticostriatal abnormalities, encompassing the abnormal amplitude of spontaneous glutamatergic transmission and the loss of Long Term Potentiation (LTP) in Tph2(GFP/GFP) mice of both sexes, while this form of plasticity is normally expressed in control mice (Tph2(+/+)). Once LTP is induced, only the Tph2(+/GFP) female mice present a loss of synaptic depotentiation. Conclusion: We showed a relevant role of the interaction between dopaminergic and serotonergic systems in controlling striatal synaptic plasticity. Overall, our data unveil that 5-HT plays a primary role in regulating DA-dependent corticostriatal plasticity in a sex-related manner and propose altered 5-HT levels as a critical determinant of disease-associated plasticity defects.

Despite recent progresses in robotic rehabilitation technologies, their efficacy for post-stroke motor recovery is still limited. Such limitations might stem from the insufficient enhancement of plasticity mechanisms, crucial for functional recovery. Here, we designed a clinically relevant strategy that combines robotic rehabilitation with chemogenetic stimulation of serotonin release to boost plasticity. These two approaches acted synergistically to enhance post-stroke motor performance. Indeed, mice treated with our combined therapy showed substantial functional gains that persisted beyond the treatment period and generalized to non-trained tasks. Motor recovery was associated with a reduction in electrophysiological and neuroanatomical markers of GABAergic neurotransmission, suggesting disinhibition in perilesional areas. To unveil the translational potentialities of our approach, we specifically targeted the serotonin 1A receptor by delivering Buspirone, a clinically approved drug, in stroke mice undergoing robotic rehabilitation. Administration of Buspirone restored motor impairments similarly to what observed with chemogenetic stimulation, showing the immediate translational potential of this combined approach to significantly improve motor recovery after stroke.

Mutations in the synaptic scaffolding protein SHANK3 are a major cause of autism and are associated with prominent intellectual and language deficits. However, the neural mechanisms whereby SHANK3 deficiency affects higher-order socio-communicative functions remain unclear. Using high-resolution functional and structural MRI in adult male mice, here we show that loss of Shank3 (Shank3B−/−) results in disrupted local and long-range prefrontal and frontostriatal functional connectivity. We document that prefrontal hypoconnectivity is associated with reduced short-range cortical projections density, and reduced gray matter volume. Finally, we show that prefrontal disconnectivity is predictive of social communication deficits, as assessed with ultrasound vocalization recordings. Collectively, our results reveal a critical role of SHANK3 in the development of prefrontal anatomy and function, and suggest that SHANK3 deficiency may predispose to intellectual disability and socio-communicative impairments via dysregulation of higher-order cortical connectivity. SIGNIFICANCE STATEMENT Mutations in the synaptic scaffolding protein SHANK3 are commonly associated with autism, intellectual, and language deficits. Previous research has linked SHANK3 deficiency to basal ganglia dysfunction, motor stereotypies, and social deficits. However, the neural mechanism whereby Shank3 gene mutations affects cortical functional connectivity and higher-order socio-communicative functions remain unclear. Here we show that loss of SHANK3 in mice results in largely disrupted functional connectivity and abnormal gray matter anatomy in prefrontal areas. We also show that prefrontal connectivity disruption is tightly linked to socio-communicative deficits. Our findings suggest that SHANK3 is a critical orchestrator of frontocortical function, and that disrupted connectivity of prefrontal areas may underpin socio-communicative impairments observed in SHANK3 mutation carriers.

Currently available inhibitory optogenetic tools provide short and transient silencing of neurons, but they cannot provide long-lasting inhibition because of the requirement for high light intensities. Here we present an optimized blue-light-sensitive synthetic potassium channel, BLINK2, which showed good expression in neurons in three species. The channel is activated by illumination with low doses of blue light, and in our experiments it remained active over (tens of) minutes in the dark after the illumination was stopped. This activation caused long periods of inhibition of neuronal firing in ex vivo recordings of mouse neurons and impaired motor neuron response in zebrafish in vivo. As a proof-of-concept application, we demonstrated that in a freely moving rat model of neuropathic pain, the activation of a small number of BLINK2 channels caused a long-lasting (>30 min) reduction in pain sensation.

Functional connectivity aberrancies, as measured with resting-state fMRI (rsfMRI), have been consistently observed in the brain of autism spectrum disorders (ASD) patients. However, the genetic and neurobiological underpinnings of these findings remain unclear. Homozygous mutations in Contactin Associated Protein-like 2 (CNTNAP2), a neurexin-related cell-adhesion protein, are strongly linked to autism and epilepsy. Here we used rsfMRI to show that homozygous mice lacking Cntnap2 exhibit reduced long-range and local functional connectivity in prefrontal and midline brain “connectivity hubs”. Long-range rsfMRI connectivity impairments affected heteromodal cortical regions and were prominent between fronto-posterior components of the mouse default-mode network (DMN), an effect that was associated with reduced social investigation, a core “autism trait” in mice. Notably, viral tracing revealed reduced frequency of prefrontal-projecting neural clusters in the cingulate cortex of Cntnap2 −/− mutants, suggesting a possible contribution of defective mesoscale axonal wiring to the observed functional impairments. Macroscale cortico-cortical white matter organization appeared to be otherwise preserved in these animals. These findings reveal a key contribution of ASD-associated gene CNTNAP2 in modulating macroscale functional connectivity, and suggest that homozygous loss-of-function mutations in this gene may predispose to neurodevelopmental disorders and autism through a selective dysregulation of connectivity in integrative prefrontal areas.

Serotonin (5-HT)-synthetizing neurons, which are confined in the raphe nuclei of the rhombencephalon, provide a pervasive innervation of the central nervous system (CNS) and are involved in the modulation of a plethora of functions in both developing and adult brain. Classical studies have described the post-natal development of serotonergic axons as a linear process of terminal field innervation. However, technical limitations have hampered a fine morphological characterization. With the advent of genetic mouse models, the possibility to label specific neuronal populations allowed the rigorous measurement of their axonal morphological features as well as their developmental dynamics. Here, we used the Tph2GFP knock-in mouse line, in which GFP expression allows punctual identification of serotonergic neurons and axons, for confocal microscope imaging and we performed 3-dimensional reconstruction in order to morphologically characterize the development of serotonergic fibers in specified brain targets from birth to adulthood. Our analysis highlighted region-specific developmental patterns of serotonergic fiber density ranging from a linear and progressive colonization of the target (Caudate/Putamen, Basolateral Amygdala, Geniculate Nucleus and Substantia Nigra) to a transient increase in fiber density (medial Prefrontal Cortex, Globus Pallidus, Somatosensory Cortex and Hippocampus) occurring with a region-specific timing. Despite a common pattern of early post-natal morphological maturation in which a progressive rearrangement from a dot-shaped to a regular and smooth fiber morphology was observed, starting from post-natal day 28 serotonergic fibers acquire the region specific morphological features present in the adult. In conclusion, we provided novel, target-specific insights on the morphology and temporal dynamics of the developing serotonergic fibers.