Differential expression of paralog RNA binding proteins establishes a dynamic splicing program required for normal cerebral cortex development.

Sam68 and SLM2 are paralog RNA binding proteins (RBPs) expressed in the cerebral cortex and display similar splicing activities. However, their relative functions during cortical development are unknown. We found that these RBPs exhibit an opposite expression pattern during development. Sam68 expression declines postnatally while SLM2 increases after birth, and this developmental pattern is reinforced by hierarchical control of Sam68 expression by SLM2. Analysis of Sam68:Slm2 double knockout (Sam68:Slm2dko) mice revealed hundreds of exons that respond to joint depletion of these proteins. Moreover, parallel analysis of single and double knockout cortices indicated that exons regulated mainly by SLM2 are characterized by a dynamic splicing pattern during development, whereas Sam68-dependent exons are spliced at relatively constant rates. Dynamic splicing of SLM2-sensitive exons is completely suppressed in the Sam68:Slm2dko developing cortex. Sam68:Slm2dko mice die perinatally with defects in neurogenesis and in neuronal differentiation, and develop a hydrocephalus, consistent with splicing alterations in genes related to these biological processes. Thus, our study reveals that developmental control of separate Sam68 and Slm2 paralog genes encoding homologous RBPs enables the orchestration of a dynamic splicing program needed for brain development and viability, while ensuring a robust redundant mechanism that supports proper cortical development.

Astrocyte Responses Influence Local Effects of Whole-Brain Magnetic Stimulation in Parkinsonian Rats.

BACKGROUND: Excessive glutamatergic transmission in the striatum is implicated in Parkinson's disease (PD) progression. Astrocytes maintain glutamate homeostasis, protecting from excitotoxicity through the glutamate-aspartate transporter (GLAST), whose alterations have been reported in PD. Noninvasive brain stimulation using intermittent theta-burst stimulation (iTBS) acts on striatal neurons and glia, inducing neuromodulatory effects and functional recovery in experimental parkinsonism. OBJECTIVE: Because PD is associated with altered astrocyte function, we hypothesized that acute iTBS, known to rescue striatal glutamatergic transmission, exerts regional- and cell-specific effects through modulation of glial functions. METHODS: 6-Hydroxydopamine-lesioned rats were exposed to acute iTBS, and the areas predicted to be more responsive by a biophysical, hyper-realistic computational model that faithfully reconstructs the experimental setting were analyzed. The effects of iTBS on glial cells and motor behavior were evaluated by molecular and morphological analyses, and CatWalk and Stepping test, respectively. RESULTS: As predicted by the model, the hippocampus, cerebellum, and striatum displayed a marked c-FOS activation after iTBS, with the striatum showing specific morphological and molecular changes in the astrocytes, decreased phospho-CREB levels, and recovery of GLAST. Striatal-dependent motor performances were also significantly improved. CONCLUSION: These data uncover an unknown iTBS effect on astrocytes, advancing the understanding of the complex mechanisms involved in TMS-mediated functional recovery. Data on numerical dosimetry, obtained with a degree of anatomical details never before considered and validated by the biological findings, provide a framework to predict the electric-field induced in different specific brain areas and associate it with functional and molecular changes. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

GABAergic miR-34a regulates Dorsal Raphè inhibitory transmission in response to aversive, but not rewarding, stimuli.

The brain employs distinct circuitries to encode positive and negative valence stimuli, and dysfunctions of these neuronal circuits have a key role in the etiopathogenesis of many psychiatric disorders. The Dorsal Raphè Nucleus (DRN) is involved in various behaviors and drives the emotional response to rewarding and aversive experiences. Whether specific subpopulations of neurons within the DRN encode these behaviors with different valence is still unknown. Notably, microRNA expression in the mammalian brain is characterized by tissue and neuronal specificity, suggesting that it might play a role in cell and circuit functionality. However, this specificity has not been fully exploited. Here, we demonstrate that microRNA-34a (miR-34a) is selectively expressed in a subpopulation of GABAergic neurons of the ventrolateral DRN. Moreover, we report that acute exposure to both aversive (restraint stress) and rewarding (chocolate) stimuli reduces GABA release in the DRN, an effect prevented by the inactivation of DRN miR-34a or its genetic deletion in GABAergic neurons in aversive but not rewarding conditions. Finally, miR-34a inhibition selectively reduced passive coping with severe stressors. These data support a role of miR-34a in regulating GABAergic neurotransmitter activity and behavior in a context-dependent manner and suggest that microRNAs could represent a functional signature of specific neuronal subpopulations with valence-specific activity in the brain.

Intensive exercise ameliorates motor and cognitive symptoms in experimental Parkinson's disease restoring striatal synaptic plasticity.

Intensive physical activity improves motor functions in patients with Parkinson's disease (PD) at early stages. However, the mechanisms underlying the beneficial effects of exercise on PD-associated neuronal alterations have not been fully clarified yet. Here, we tested the hypothesis that an intensive treadmill training program rescues alterations in striatal plasticity and early motor and cognitive deficits in rats receiving an intrastriatal injection of alpha-synuclein (α-syn) preformed fibrils. Improved motor control and visuospatial learning in active animals were associated with a recovery of dendritic spine density alterations and a lasting rescue of a physiological corticostriatal long-term potentiation (LTP). Pharmacological analyses of LTP show that modulations of N-methyl-d-aspartate receptors bearing GluN2B subunits and tropomyosin receptor kinase B, the main brain-derived neurotrophic factor receptor, are involved in these beneficial effects. We demonstrate that intensive exercise training has effects on the early plastic alterations induced by α-syn aggregates and reduces the spread of toxic α-syn species to other vulnerable brain areas.

SEMA6C: a novel adhesion-independent FAK and YAP activator, required for cancer cell viability and growth.

Transmembrane semaphorins are signaling molecules, controlling axonal wiring and embryo development, which are increasingly implicated in human diseases. Semaphorin 6C (Sema6C) is a poorly understood family member and its functional role is still unclear. Upon targeting Sema6C expression in a range of cancer cells, we observed dramatic growth suppression, decreased ERK phosphorylation, upregulation of cell cycle inhibitor proteins p21, p27 and p53, and the onset of cell senescence, associated with activation of autophagy. These data are consistent with a fundamental requirement for Sema6C to support viability and growth in cancer cells. Mechanistically, we unveiled a novel signaling pathway elicited by Sema6C, and dependent on its intracellular domain, mediated by tyrosine kinases c-Abl and Focal Adhesion Kinase (FAK). Sema6C was found in complex with c-Abl, and induced its phosphorylation, which in turn led to FAK activation, independent of cell-matrix adhesion. Sema6C-induced FAK activity was furthermore responsible for increased nuclear localization of YAP transcriptional regulator. Moreover, Sema6C conferred YAP signaling-dependent long-term cancer cell survival upon nutrient deprivation. In conclusion, our findings demonstrate that Sema6C elicits a cancer promoting-signaling pathway sustaining cell viability and self-renewal, independent of growth factors and nutrients availability.

Myelin basic protein recovery during PKU mice lifespan and the potential role of microRNAs on its regulation.

Untreated phenylketonuria (PKU) patients and PKU animal models show hypomyelination in the central nervous system and white matter damages, which are accompanied by myelin basic protein (MBP) impairment. Despite many assumptions, the primary explanation of the mentioned cerebral outcomes remains elusive. In this study, MBP protein and mRNA expression on brains of wild type (WT) and phenylketonuric (ENU2) mice were analyzed throughout mice lifespan (14-60-180-270-360-540 post-natal days, PND). The results confirmed the low MBP expression at first PND times, while revealed an unprecedented progressive MBP protein expression recovery in aged ENU2 mice. Unexpectedly, unaltered MBP mRNA expression between WT and ENU2 was always observed. Additionally, for the same time intervals, a significant decrease of the phenylalanine concentration in the peripheral blood and brain of ENU2 mice was detected, to date, for the first time. In this scenario, a translational hindrance of MBP during initial and late cerebral development in ENU2 mice was hypothesized, leading to the execution of a microRNA microarray analysis on 60 PND brains, which was followed by a proteomic assay on 60 and 360 PND brains in order to validate in silico miRNA-target predictions. Taken together, miR-218-1-3p, miR-1231-3p and miR-217-5p were considered as the most impactful microRNAs, since a downregulation of their potential targets (MAG, CNTNAP2 and ANLN, respectively) can indirectly lead to a low MBP protein expression. These miRNAs, in addition, follow an opposite expression trend compared to MBP during adulthood, and their target proteins revealed a complete normalization in aged ENU2 mice. In conclusion, these results provide a new perspective on the PKU pathophysiology understanding and on a possible treatment, emphasizing the potential modulating role of differentially expressed microRNAs in MBP expression on PKU brains during PKU mouse lifespan.

Upregulation of Ca2+-binding proteins contributes to VTA dopamine neuron survival in the early phases of Alzheimer's disease in Tg2576 mice.

BACKGROUND: Recent clinical and experimental studies have highlighted the involvement of Ventral Tegmental Area (VTA) dopamine (DA) neurons for the early pathogenesis of Alzheimer's Disease (AD). We have previously described a progressive and selective degeneration of these neurons in the Tg2576 mouse model of AD, long before amyloid-beta plaque formation. The degenerative process in DA neurons is associated with an autophagy flux impairment, whose rescue can prevent neuronal loss. Impairments in autophagy can be the basis for accumulation of damaged mitochondria, leading to disturbance in calcium (Ca2+) homeostasis, and to functional and structural deterioration of DA neurons. METHODS: In Tg2576 mice, we performed amperometric recordings of DA levels and analysis of dopaminergic fibers in the Nucleus Accumbens - a major component of the ventral striatum precociously affected in AD patients - together with retrograde tracing, to identify the most vulnerable DA neuron subpopulations in the VTA. Then, we focused on these neurons to analyze mitochondrial integrity and Apoptosis-inducing factor (AIF) localization by electron and confocal microscopy, respectively. Stereological cell count was also used to evaluate degeneration of DA neuron subpopulations containing the Ca2+-binding proteins Calbindin-D28K and Calretinin. The expression levels for these proteins were analyzed by western blot and confocal microscopy. Lastly, using electrophysiology and microfluorometry we analyzed VTA DA neuron intrinsic properties and cytosolic free Ca2+ levels. RESULTS: We found a progressive degeneration of mesolimbic DA neurons projecting to the ventral striatum, located in the paranigral nucleus and parabrachial pigmented subnucleus of the VTA. At the onset of degeneration (3 months of age), the vulnerable DA neurons in the Tg2576 accumulate damaged mitochondria, while AIF translocates from the mitochondria to the nucleus. Although we describe an age-dependent loss of the DA neurons expressing Calbindin-D28K or Calretinin, we observed that the remaining cells upregulate the levels of Ca2+-binding proteins, and the free cytosolic levels of Ca2+ in these neurons are significantly decreased. Coherently, TUNEL-stained Tg2576 DA neurons express lower levels of Calbindin-D28K when compared with non-apoptotic cells. CONCLUSION: Overall, our results suggest that the overexpression of Ca2+-binding proteins in VTA DA neurons might be an attempt of cells to survive by increasing their ability to buffer free Ca2+. Exploring strategies to overexpress Ca2+-binding proteins could be fundamental to reduce neuronal suffering and improve cognitive and non-cognitive functions in AD.

Correction: Cutuli et al. Neuroprotective Role of Dietary Supplementation with Omega-3 Fatty Acids in the Presence of Basal Forebrain Cholinergic Neurons Degeneration in Aged Mice. Int. J. Mol. Sci. 2020, 21, 1741.

The authors wish to make the following corrections to this paper [...].

Dynamic Interactions between Tumor Cells and Brain Microvascular Endothelial Cells in Glioblastoma.

GBM is the most aggressive brain tumor among adults. It is characterized by extensive vascularization, and its further growth and recurrence depend on the formation of new blood vessels. In GBM, tumor angiogenesis is a multi-step process involving the proliferation, migration and differentiation of BMECs under the stimulation of specific signals derived from the cancer cells through a wide variety of communication routes. In this review, we discuss the dynamic interaction between BMECs and tumor cells by providing evidence of how tumor cells hijack the BMECs for the formation of new vessels. Tumor cell-BMECs interplay involves multiple routes of communication, including soluble factors, such as chemokines and cytokines, direct cell-cell contact and extracellular vesicles that participate in and fuel this cooperation. We also describe how this interaction is able to modify the BMECs structure, metabolism and physiology in a way that favors tumor growth and invasiveness. Finally, we briefly reviewed the recent advances and the potential future implications of some high-throughput 3D models to better understanding the complexity of BMECs-tumor cell interaction.

Restoration of ER proteostasis attenuates remote apoptotic cell death after spinal cord injury by reducing autophagosome overload.

The pathogenic mechanisms that underlie the progression of remote degeneration after spinal cord injury (SCI) are not fully understood. In this study, we examined the relationship between endoplasmic reticulum (ER) stress and macroautophagy, hereafter autophagy, and its contribution to the secondary damage and outcomes that are associated with remote degeneration after SCI. Using a rat model of spinal cord hemisection at the cervical level, we measured ER stress and autophagy markers in the axotomized neurons of the red nucleus (RN). In SCI animals, mRNA and protein levels of markers of ER stress, such as GRP78, CHOP, and GADD34, increased 1 day after the injury, peaking on Day 5. Notably, in SCI animals, the increase of ER stress markers correlated with a blockade in autophagic flux, as evidenced by the increase in microtubule-associated protein 2 light chain 3 (LC3-II) and p62/SQSTM1 (p62) and the decline in LAMP1 and LAMP2 levels. After injury, treatment with guanabenz protected neurons from UPR failure and increased lysosomes biogenesis, unblocking autophagic flux. These effects correlated with greater activation of TFEB and improved neuronal survival and functional recovery-effects that persisted after suspension of the treatment. Collectively, our results demonstrate that in remote secondary damage, impairments in autophagic flux are intertwined with ER stress, an association that contributes to the apoptotic cell death and functional damage that are observed after SCI.

Early Life Social Stress Causes Sex- and Region-Dependent Dopaminergic Changes that Are Prevented by Minocycline.

Early life stress (ELS) is known to modify trajectories of brain dopaminergic development, but the mechanisms underlying have not been determined. ELS perturbs immune system and microglia reactivity, and inflammation and microglia influence dopaminergic transmission and development. Whether microglia mediate the effects of ELS on dopamine (DA) system development is still unknown. We explored the effects of repeated early social stress on development of the dopaminergic system in male and female mice through histological, electrophysiological, and transcriptomic analyses. Furthermore, we tested whether these effects could be mediated by ELS-induced altered microglia/immune activity through a pharmacological approach. We found that social stress in early life altered DA neurons morphology, reduced dopamine transporter (DAT) and tyrosine hydroxylase expression, and lowered DAT-mediated currents in the ventral tegmental area but not substantia nigra of male mice only. Notably, stress-induced DA alterations were prevented by minocycline, an inhibitor of microglia activation. Transcriptome analysis in the developing male ventral tegmental area revealed that ELS caused downregulation of dopaminergic transmission and alteration in hormonal and peptide signaling pathways. Results from this study offer new insight into the mechanisms of stress response and altered brain dopaminergic maturation after ELS, providing evidence of neuroimmune interaction, sex differences, and regional specificity.

Interleukin-17 affects synaptic plasticity and cognition in an experimental model of multiple sclerosis.

Cognitive impairment (CI) is a disabling concomitant of multiple sclerosis (MS) with a complex and controversial pathogenesis. The cytokine interleukin-17A (IL-17A) is involved in the immune pathogenesis of MS, but its possible effects on synaptic function and cognition are still largely unexplored. In this study, we show that the IL-17A receptor (IL-17RA) is highly expressed by hippocampal neurons in the CA1 area and that exposure to IL-17A dose-dependently disrupts hippocampal long-term potentiation (LTP) through the activation of its receptor and p38 mitogen-activated protein kinase (MAPK). During experimental autoimmune encephalomyelitis (EAE), IL-17A overexpression is paralleled by hippocampal LTP dysfunction. An in vivo behavioral analysis shows that visuo-spatial learning abilities are preserved when EAE is induced in mice lacking IL-17A. Overall, this study suggests a key role for the IL-17 axis in the neuro-immune cross-talk occurring in the hippocampal CA1 area and its potential involvement in synaptic dysfunction and MS-related CI.

Transcranial Magnetic Stimulation Exerts "Rejuvenation" Effects on Corticostriatal Synapses after Partial Dopamine Depletion.

BACKGROUND: In experimental models of Parkinson's disease (PD), different degrees of degeneration to the nigrostriatal pathway produce distinct profiles of synaptic alterations that depend on progressive changes in N-methyl-D-aspartate receptors (NMDAR)-mediated functions. Repetitive transcranial magnetic stimulation (rTMS) induces modifications in glutamatergic and dopaminergic systems, suggesting that it may have an impact on glutamatergic synapses modulated by dopamine neurotransmission. However, no studies have so far explored the mechanisms of rTMS effects at early stages of PD. OBJECTIVES: We tested the hypothesis that in vivo application of rTMS with intermittent theta-burst stimulation (iTBS) pattern alleviates corticostriatal dysfunctions by modulating NMDAR-dependent plasticity in a rat model of early parkinsonism. METHODS: Dorsolateral striatal spiny projection neurons (SPNs) activity was studied through ex vivo whole-cell patch-clamp recordings in corticostriatal slices obtained from 6-hydroxydopamine-lesioned rats, subjected to a single session (acute) of iTBS and tested for forelimb akinesia with the stepping test. Immunohistochemical analyses were performed to analyze morphological correlates of plasticity in SPNs. RESULTS: Acute iTBS ameliorated limb akinesia and rescued corticostriatal long-term potentiation (LTP) in SPNs of partially lesioned rats. This effect was abolished by applying a selective inhibitor of GluN2B-subunit-containing NMDAR, suggesting that iTBS treatment could be associated with an enhanced activation of specific NMDAR subunits, which are major regulators of structural plasticity during synapse development. Morphological analyses of SPNs revealed that iTBS treatment reverted dendritic spine loss inducing a prevalence of thin-elongated spines in the biocytin-filled SPNs. CONCLUSIONS: Taken together, our data identify that an acute iTBS treatment produces a series of plastic changes underlying striatal compensatory adaptation in the parkinsonian basal ganglia circuit. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Early life stress exposure worsens adult remote microglia activation, neuronal death, and functional recovery after focal brain injury.

Trauma to the central nervous system (CNS) is a devastating condition resulting in severe functional impairments that strongly vary among patients. Patients' features, such as age, social and cultural environment, and pre-existing psychiatric conditions may be particularly relevant for determining prognosis after CNS trauma. Although several studies demonstrated the impact of adult psycho-social stress exposure on functional recovery after CNS damage, no data exist regarding the long-term effects of the exposure to such experience at an early age. Here, we assessed whether early life stress (ELS) hampers the neuroinflammatory milieuand the functional recovery after focal brain injury in adulthood by using a murine model of ELS exposure combined with hemicerebellectomy (HCb), a model of remote damage. We found that ELS permanently altered microglia responses such that, once experienced HCb, they produced an exaggerated remote inflammatory response - consistent with a primed phenotype - associated with increased cell death and worse functional recovery. Notably, prevention of microglia/macrophages activation by GW2580 treatment during ELS exposure significantly reduced microglia responses, cell death and improved functional recovery. Conversely, GW2580 treatment administered in adulthood after HCb was ineffective in reducing inflammation and cell death or improving functional recovery. Our findings highlight that ELS impacts the immune system maturation producing permanent changes, and that it is a relevant factor modulating the response to a CNS damage. Further studies are needed to clarify the mechanisms underlying the interaction between ELS and brain injury with the aim of developing targeted treatments to improve functional recovery after CNS damage.

Nilotinib restores memory function by preventing dopaminergic neuron degeneration in a mouse model of Alzheimer's Disease.

What happens precociously to the brain destined to develop Alzheimer's Disease (AD) still remains to be elucidated and this is one reason why effective AD treatments are missing. Recent experimental and clinical studies indicate that the degeneration of the dopaminergic (DA) neurons in the Ventral Tegmental Area (VTA) could be one of the first events occurring in AD. However, the causes of the increased vulnerability of DA neurons in AD are missing. Here, we deeply investigate the physiology of DA neurons in the VTA before, at the onset, and after onset of VTA neurodegeneration. We use the Tg2576 mouse model of AD, overexpressing a mutated form of the human APP, to identify molecular targets that can be manipulated pharmacologically. We show that in Tg2576 mice, DA neurons of the VTA at the onset of degeneration undergo slight but functionally relevant changes in their electrophysiological properties and cell morphology. Importantly, these changes are associated with accumulation of autophagosomes, suggestive of a dysfunctional autophagy, and with enhanced activation of c-Abl, a tyrosine kinase previously implicated in the pathogenesis of neurodegenerative diseases. Chronic treatment of Tg2576 mice with Nilotinib, a validated c-Abl inhibitor, reduces c-Abl phosphorylation, improves autophagy, reduces Aß levels and - more importantly - prevents degeneration as well as functional and morphological alterations in DA neurons of the VTA. Interestingly, the drug prevents the reduction of DA outflow to the hippocampus and ameliorates hippocampal-related cognitive functions. Our results strive to identify early pathological brain changes in AD, to provide a rational basis for new therapeutic interventions able to slow down the disease progression.

Exposure to different early-life stress experiences results in differentially altered DNA methylation in the brain and immune system.

The existence of a proportional relationship between the number of early-life stress (ELS) events experienced and the impoverishment of child mental health has been hypothesized. However, different types of ELS experiences may be associated with different neuro-psycho-biological impacts, due to differences in the intrinsic nature of the stress. DNA methylation is one of the molecular mechanisms that have been implicated in the "translation" of ELS exposure into neurobiological and behavioral abnormalities during adulthood. Here, we investigated whether different ELS experiences resulted in differential impacts on global DNA methylation levels in the brain and blood samples from mice and humans. ELS exposure in mice resulted in observable changes in adulthood, with exposure to social isolation inducing more dramatic alterations in global DNA methylation levels in several brain structures compared with exposure to a social threatening environment. Moreover, these two types of stress resulted in differential impacts on the epigenetic programming of different brain regions and cellular populations, namely microglia. In a pilot clinical study, blood global DNA methylation levels and exposure to childhood neglect or abuse were investigated in patients presenting with major depressive disorder or substance use disorder. A significant effect of the mental health diagnosis on global methylation levels was observed, but no effect of either childhood abuse or neglect was detected. These findings demonstrate that different types of ELS have differential impacts on epigenetic programming, through DNA methylation in specific brain regions, and that these differential impacts are associated with the different behavioral outcomes observed after ELS experiences.

Neuroprotective Role of Dietary Supplementation with Omega-3 Fatty Acids in the Presence of Basal Forebrain Cholinergic Neurons Degeneration in Aged Mice.

As major components of neuronal membranes, omega-3 polyunsaturated fatty acids (n-3 PUFA) exhibit a wide range of regulatory functions. Recent human and animal studies indicate that n-3 PUFA may exert beneficial effects on aging processes. Here we analyzed the neuroprotective influence of n-3 PUFA supplementation on behavioral deficits, hippocampal neurogenesis, volume loss, and astrogliosis in aged mice that underwent a selective depletion of basal forebrain cholinergic neurons. Such a lesion represents a valid model to mimic a key component of the cognitive deficits associated with dementia. Aged mice were supplemented with n-3 PUFA or olive oil (as isocaloric control) for 8 weeks and then cholinergically depleted with mu-p75-saporin immunotoxin. Two weeks after lesioning, mice were behaviorally tested to assess anxious, motivational, social, mnesic, and depressive-like behaviors. Subsequently, morphological and biochemical analyses were performed. In lesioned aged mice the n-3 PUFA pre-treatment preserved explorative skills and associative retention memory, enhanced neurogenesis in the dentate gyrus, and reduced volume and VAChT levels loss as well as astrogliosis in hippocampus. The present findings demonstrating that n-3 PUFA supplementation before cholinergic depletion can counteract behavioral deficits and hippocampal neurodegeneration in aged mice advance a low-cost, non-invasive preventive tool to enhance life quality during aging.