Gut microbiota promotes pain chronicity in Myosin1A deficient male mice.

Chronic pain is a heavily debilitating condition and a huge socio-economic burden, with no efficient treatment. Over the past decade, the gut microbiota has emerged as an important regulator of nervous system's health and disease states. Yet, its contribution to the pathogenesis of chronic somatic pain remains poorly documented. Here, we report that male but not female mice lacking Myosin1a (KO) raised under single genotype housing conditions (KO-SGH) are predisposed to develop chronic pain in response to a peripheral tissue injury. We further underscore the potential of MYO1A loss-of-function to alter the composition of the gut microbiota and uncover a functional connection between the vulnerability to chronic pain and the dysbiotic gut microbiota of KO-SGH males. As such, parental antibiotic treatment modifies gut microbiota composition and completely rescues the injury-induced pain chronicity in male KO-SGH offspring. Furthermore, in KO-SGH males, this dysbiosis is accompanied by a transcriptomic activation signature in the dorsal root ganglia (DRG) macrophage compartment, in response to tissue injury. We identify CD206+CD163- and CD206+CD163+ as the main subsets of DRG resident macrophages and show that both are long-lived and self-maintained and exhibit the capacity to monitor the vasculature. Consistently, in vivo depletion of DRG macrophages rescues KO-SGH males from injury-induced chronic pain underscoring a deleterious role for DRG macrophages in a Myo1a-loss-of function context. Together, our findings reveal gene-sex-microbiota interactions in determining the predisposition to injury-induced chronic pain and point-out DRG macrophages as potential effector cells.

In vivo magnetic resonance spectroscopy in the brain of Cdkl5 null mice reveals a metabolic profile indicative of mitochondrial dysfunctions.

Mutations in the X-linked CDKL5 gene cause CDKL5 deficiency disorder (CDD), a severe neurodevelopmental condition mainly characterized by infantile epileptic encephalopathy, intellectual disability, and autistic features. The molecular mechanisms underlying the clinical symptoms remain largely unknown and the identification of reliable biomarkers in animal models will certainly contribute to increase our comprehension of CDD as well as to assess the efficacy of therapeutic strategies. Here, we used different Magnetic Resonance (MR) methods to disclose structural, functional, or metabolic signatures of Cdkl5 deficiency in the brain of adult mice. We found that loss of Cdkl5 does not cause cerebral atrophy but affects distinct brain areas, particularly the hippocampus. By in vivo proton-MR spectroscopy (MRS), we revealed in the Cdkl5 null brain a metabolic dysregulation indicative of mitochondrial dysfunctions. Accordingly, we unveiled a significant reduction in ATP levels and a decrease in the expression of complex IV of mitochondrial electron transport chain. Conversely, the number of mitochondria appeared preserved. Importantly, we reported a significant defect in the activation of one of the major regulators of cellular energy balance, the adenosine monophosphate-activated protein kinase (AMPK), that might contribute to the observed metabolic impairment and become an interesting therapeutic target for future preclinical trials. In conclusion, MRS revealed in the Cdkl5 null brain the presence of a metabolic dysregulation suggestive of a mitochondrial dysfunction that permitted to foster our comprehension of Cdkl5 deficiency and brought our interest towards targeting mitochondria as therapeutic strategy for CDD.

Neurochemical and Ultrastructural Characterization of Unmyelinated Non-peptidergic C-Nociceptors and C-Low Threshold Mechanoreceptors Projecting to Lamina II of the Mouse Spinal Cord.

C-nociceptors (C-Ncs) and non-nociceptive C-low threshold mechanoreceptors (C-LTMRs) are two subpopulations of small unmyelinated non-peptidergic C-type neurons of the dorsal root ganglia (DRGs) with central projections displaying a specific pattern of termination in the spinal cord dorsal horn. Although these two subpopulations exist in several animals, remarkable neurochemical differences occur between mammals, particularly rat/humans from one side and mouse from the other. Mouse is widely investigated by transcriptomics. Therefore, we here studied the immunocytochemistry of murine C-type DRG neurons and their central terminals in spinal lamina II at light and electron microscopic levels. We used a panel of markers for peptidergic (CGRP), non-peptidergic (IB4), nociceptive (TRPV1), non-nociceptive (VGLUT3) C-type neurons and two strains of transgenic mice: the TAFA4Venus knock-in mouse to localize the TAFA4+ C-LTMRs, and a genetically engineered ginip mouse that allows an inducible and tissue-specific ablation of the DRG neurons expressing GINIP, a key modulator of GABABR-mediated analgesia. We confirmed that IB4 and TAFA4 did not coexist in small non-peptidergic C-type DRG neurons and separately tagged the C-Ncs and the C-LTMRs. We then showed that TRPV1 was expressed in only about 7% of the IB4+ non-peptidergic C-Ncs and their type Ia glomerular terminals within lamina II. Notably, the selective ablation of GINIP did not affect these neurons, whereas it reduced IB4 labeling in the medial part of lamina II and the density of C-LTMRs glomerular terminals to about one half throughout the entire lamina. We discuss the significance of these findings for interspecies differences and functional relevance.

Cytoarchitectural analysis of the neuron-to-glia association in the dorsal root ganglia of normal and diabetic mice.

Dorsal root ganglia (DRGs) host the somata of sensory neurons which convey information from the periphery to the central nervous system. These neurons have heterogeneous size and neurochemistry, and those of small-to-medium size, which play an important role in nociception, form two distinct subpopulations based on the presence (peptidergic) or absence (non-peptidergic) of transmitter neuropeptides. Few investigations have so far addressed the spatial relationship between neurochemically different subpopulations of DRG neurons and glia. We used a whole-mount mouse lumbar DRG preparation, confocal microscopy and computer-aided 3D analysis to unveil that IB4+ non-peptidergic neurons form small clusters of 4.7 ± 0.26 cells, differently from CGRP+ peptidergic neurons that are, for the most, isolated (1.89 ± 0.11 cells). Both subpopulations of neurons are ensheathed by a thin layer of satellite glial cells (SGCs) that can be observed after immunolabeling with the specific marker glutamine synthetase (GS). Notably, at the ultrastructural level we observed that this glial layer was discontinuous, as there were patches of direct contact between the membranes of two adjacent IB4+ neurons. To test whether this cytoarchitectonic organization was modified in the diabetic neuropathy, one of the most devastating sensory pathologies, mice were made diabetic by streptozotocin (STZ). In diabetic animals, cluster organization of the IB4+ non-peptidergic neurons was maintained, but the neuro-glial relationship was altered, as STZ treatment caused a statistically significant increase of GS staining around CGRP+ neurons but a reduction around IB4+ neurons. Ultrastructural analysis unveiled that SGC coverage was increased at the interface between IB4+ cluster-forming neurons in diabetic mice, with a 50% reduction in the points of direct contacts between cells. These observations demonstrate the existence of a structural plasticity of the DRG cytoarchitecture in response to STZ.

Impaired chromaffin cell excitability and exocytosis in autistic Timothy syndrome TS2-neo mouse rescued by L-type calcium channel blockers.

KEY POINTS: Tymothy syndrome (TS) is a multisystem disorder featuring cardiac arrhythmias, autism and adrenal gland dysfunction that originates from a de novo point mutation in the gene encoding the Cav1.2 (CACNA1C) L-type channel. To study the role of Cav1.2 channel signals in autism, the autistic TS2-neo mouse has been generated bearing the G406R point-mutation associated with TS type-2. Using heterozygous TS2-neo mice, we report that the G406R mutation reduces the rate of inactivation and shifts leftward the activation and inactivation of L-type channels, causing marked increase of resting Ca2+ influx ('window' Ca2+ current). The increased 'window current' causes marked reduction of NaV channel density, switches normal tonic firing to abnormal burst firing, reduces mitochondrial metabolism, induces cell swelling and decreases catecholamine release. Overnight incubations with nifedipine rescue NaV channel density, normal firing and the quantity of catecholamine released. We provide evidence that chromaffin cell malfunction derives from altered Cav1.2 channel gating. ABSTRACT: L-type voltage-gated calcium (Cav1) channels have a key role in long-term synaptic plasticity, sensory transduction, muscle contraction and hormone release. A point mutation in the gene encoding Cav1.2 (CACNA1C) causes Tymothy syndrome (TS), a multisystem disorder featuring cardiac arrhythmias, autism spectrum disorder (ASD) and adrenal gland dysfunction. In the more severe type-2 form (TS2), the missense mutation G406R is on exon 8 coding for the IS6-helix of the Cav1.2 channel. The mutation causes reduced inactivation and induces autism. How this occurs and how Cav1.2 gating-changes alter cell excitability, neuronal firing and hormone release on a molecular basis is still largely unknown. Here, using the TS2-neo mouse model of TS we show that the G406R mutation altered excitability and reduced secretory activity in adrenal chromaffin cells (CCs). Specifically, the TS2 mutation reduced the rate of voltage-dependent inactivation and shifted leftward the activation and steady-state inactivation of L-type channels. This markedly increased the resting 'window' Ca2+ current that caused an increased percentage of CCs undergoing abnormal action potential (AP) burst firing, cell swelling, reduced mitochondrial metabolism and decreased catecholamine release. The increased 'window' Ca2+ current caused also decreased NaV channel density and increased steady-state inactivation, which contributed to the increased abnormal burst firing. Overnight incubation with the L-type channel blocker nifedipine rescued the normal AP firing of CCs, the density of functioning NaV channels and their steady-state inactivation. We provide evidence that CC malfunction derives from the altered Cav1.2 channel gating and that dihydropyridines are potential therapeutics for ASD.

GABAB receptors-mediated tonic inhibition of glutamate release from Aß fibers in rat laminae III/IV of the spinal cord dorsal horn.

Presynaptic GABAB receptors (GABABRs) are highly expressed in dorsal root ganglion neurons and spinal cord dorsal horn. GABABRs located in superficial dorsal horn play an important antinociceptive role, by acting at both pre- and postsynaptic sites. GABABRs expressed in deep dorsal horn could be involved in the processing of touch sensation and possibly in the generation of tactile allodynia in chronic pain. The objective of this study was to characterize the morphological and functional properties of GABABRs expressed on Aß fibers projecting to lamina III/IV and to understand their role in modulating excitatory synaptic transmission. We performed high-resolution electron microscopic analysis, showing that GABAB2 subunit is expressed on 71.9% of terminals in rat lamina III-IV. These terminals were engaged in axodendritic synapses and, for the 46%, also expressed glutamate immunoreactivity. Monosynaptic excitatory postsynaptic currents, evoked by Aß fiber stimulation and recorded from lamina III/IV neurons in spinal cord slices, were strongly depressed by application of baclofen (0.1-2.5 µM), acting as a presynaptic modulator. Application of the GABABR antagonist CGP 55845 caused, in a subpopulation of neurons, the potentiation of the first of two excitatory postsynaptic currents recorded with the paired-pulse protocol, showing that GABABRs are endogenously activated. A decrease in the paired-pulse ratio accompanied the effect of CGP 55845, implying the involvement of presynaptic GABABRs. CGP 55845 facilitated only the first excitatory postsynaptic current also during a train of four consecutive stimuli applied to Aß fibers. These results suggest that GABABRs tonically inhibit glutamate release from Aß fibers at a subset of synapses in deep dorsal horn. This modulation specifically affects only the early phase of synaptic excitation in lamina III-IV neurons.

Peripheral and central alterations affecting spinal nociceptive processing and pain at adulthood in rats exposed to neonatal maternal deprivation.

The nociceptive system of rodents is not fully developed and functional at birth. Specifically, C fibers transmitting peripheral nociceptive information establish synaptic connections in the spinal cord already during the embryonic period that only become fully functional after birth. Here, we studied the consequences of neonatal maternal deprivation (NMD, 3 h/day, P2-P12) on the functional establishment of C fiber-mediated neurotransmission in spinal cord and of pain-related behavior. In vivo recording revealed that C fiber-mediated excitation of spinal cord neurons could be observed at P14 only in control but not in NMD rats. NMD was associated with a strong alteration in the expression of growth factors controlling C nociceptor maturation as well as two-pore domain K+ channels known to set nociceptive thresholds. In good agreement, C-type sensory neurons from NMD animals appeared to be hypoexcitable but functionally connected to spinal neurons, especially those expressing TRPV1 receptors. In vivo and in vitro recordings of lamina II spinal neurons at P14 revealed that the NMD-related lack of C fiber-evoked responses resulted from an inhibitory barrage in the spinal cord dorsal horn. Eventually, C-type sensory-spinal processing could be recovered after a delay of about 10 days in NMD animals. However, animals remained hypersensitive to noxious stimulus up to P100 and this might be due to an excessive expression of Nav1.8 transcripts in DRG neurons. Together, our data provide evidence for a deleterious impact of perinatal stress exposure on the maturation of the sensory-spinal nociceptive system that may contribute to the nociceptive hypersensitivity in early adulthood.

Presynaptic modulation of spinal nociceptive transmission by glial cell line-derived neurotrophic factor (GDNF).

The role of glial cell line-derived neurotrophic factor (GDNF) in nociceptive pathways is still controversial, as both pronociceptive and antinociceptive actions have been reported. To elucidate this role in the mouse, we performed combined structural and functional studies in vivo and in acute spinal cord slices where C-fiber activation was mimicked by capsaicin challenge. Nociceptors and their terminals in superficial dorsal horn (SDH; laminae I-II) constitute two separate subpopulations: the peptidergic CGRP/somatostatin+ cells expressing GDNF and the nonpeptidergic IB4+ neurons expressing the GFRα1-RET GDNF receptor complex. Ultrastructurally the dorsal part of inner lamina II (LIIid) harbors a mix of glomeruli that either display GDNF/somatostatin (GIb)-IR or GFRα1/IB4 labeling (GIa). LIIid thus represents the preferential site for ligand-receptor interactions. Functionally, endogenous GDNF released from peptidergic CGRP/somatostatin+ nociceptors upon capsaicin stimulation exert a tonic inhibitory control on the glutamate excitatory drive of SDH neurons as measured after ERK1/2 phosphorylation assay. Real-time Ca(2+) imaging and patch-clamp experiments with bath-applied GDNF (100 nM) confirm the presynaptic inhibition of SDH neurons after stimulation of capsaicin-sensitive, nociceptive primary afferent fibers. Accordingly, the reduction of the capsaicin-evoked [Ca(2+)]i rise and of the frequency of mEPSCs in SDH neurons is specifically abolished after enzymatic ablation of GFRα1. Therefore, GDNF released from peptidergic CGRP/somatostatin+ nociceptors acutely depresses neuronal transmission in SDH signaling to nonpeptidergic IB4+ nociceptors at glomeruli in LIIid. These observations are of potential pharmacological interest as they highlight a novel modality of cross talk between nociceptors that may be relevant for discrimination of pain modalities.

Phosphorylation of histone H2AX in the mouse brain from development to senescence.

Phosphorylation of the histone H2AX (γH2AX form) is an early response to DNA damage and a marker of aging and disease in several cells and tissues outside the nervous system. Little is known about in vivo phosphorylation of H2AX in neurons, although it was suggested that γH2AX is an early marker of neuronal endangerment thus opening the possibility to target it as a neuroprotective strategy. After experimental labeling of DNA-synthesizing cells with 5-bromo-2-deoxyuridine (BrdU), we studied the brain occurrence of γH2AX in developing, postnatal, adult and senescent (2 years) mice by light and electron microscopic immunocytochemistry and Western blotting. Focal and/or diffuse γH2AX immunostaining appears in interkinetic nuclei, mitotic chromosomes, and apoptotic nuclei. Immunoreactivity is mainly associated with neurogenetic areas, i.e., the subventricular zone (SVZ) of telencephalon, the cerebellar cortex, and, albeit to a much lesser extent, the subgranular zone of the hippocampal dentate gyrus. In addition, γH2AX is highly expressed in the adult and senescent cerebral cortex, particularly the piriform cortex. Double labeling experiments demonstrate that γH2AX in neurogenetic brain areas is temporally and functionally related to proliferation and apoptosis of neuronal precursors, i.e., the type C transit amplifying cells (SVZ) and the granule cell precursors (cerebellum). Conversely, γH2AX-immunoreactive cortical neurons incorporating the S phase-label BrdU do not express the proliferation marker phosphorylated histone H3, indicating that these postmitotic cells undergo a significant DNA damage response. Our study paves the way for a better comprehension of the role of H2AX phosphorylation in the normal brain, and offers additional data to design novel strategies for the protection of neuronal precursors and mature neurons in central nervous system (CNS) degenerative diseases.

Sex-related exacerbation of injury-induced mechanical hypersensitivity in GAD67 haplodeficient mice.

ABSTRACT: Decreased GABA levels in injury-induced loss of spinal inhibition are still under intense interest and debate. Here, we show that GAD67 haplodeficient mice exhibited a prolonged injury-induced mechanical hypersensitivity in postoperative, inflammatory, and neuropathic pain models. In line with this, we found that loss of 1 copy of the GAD67-encoding gene Gad1 causes a significant decrease in GABA contents in spinal GABAergic neuronal profiles. Consequently, GAD67 haplodeficient males and females were unresponsive to the analgesic effect of diazepam. Remarkably, all these phenotypes were more pronounced in GAD67 haplodeficient females. These mice had significantly much lower amount of spinal GABA content, exhibited an exacerbated pain phenotype during the second phase of the formalin test, developed a longer lasting mechanical hypersensitivity in the chronic constriction injury of the sciatic nerve model, and were unresponsive to the pain relief effect of the GABA-transaminase inhibitor phenylethylidenehydrazine. Our study provides strong evidence for a role of GABA levels in the modulation of injury-induced mechanical pain and suggests a potential role of the GABAergic system in the prevalence of some painful diseases among females.

Extracellular Vesicles Derived from Human Liver Stem Cells Counteract Chronic Kidney Disease Development and Cardiac Dysfunction in Remnant Kidney Murine Model: The Possible Involvement of Proteases.

Fibrosis is a marker of chronic kidney disease (CKD) and consists of the accumulation of the extracellular matrix (ECM) components, causing the progressive deterioration of kidney function. Human liver stem cells (HLSCs) have anti-fibrotic activity, and HLSC-derived extracellular vesicles (EVs) mediate this effect. Herein, we evaluated the ability of HLSC-EVs to reverse renal and cardiac alterations in a murine model of partial nephrectomy (PNx) that mimics human CKD development. Furthermore, we investigated the contribution of extracellular matrix remodeling-related proteases to the anti-fibrotic effect of HLSC-EVs. PNx was performed by ligation of both poles of the left kidney, followed one week later by the removal of the right kidney. EV treatment started 4 weeks after the nephrectomy, when renal and cardiac alternations were already established, and mice were sacrificed at week eight. HLSC-EV treatment improved renal function and morphology, significantly decreasing interstitial fibrosis, glomerular sclerosis, and capillary rarefaction. This improvement was confirmed by the decreased expression of pro-fibrotic genes. Moreover, EV treatment improved cardiac function and reduced cardiac fibrosis. HLSC-EVs shuttled different proteases with ECM remodeling activity, and matrix metalloproteinase 1 (MMP-1) was involved in their anti-fibrotic effect on renal tissue. HLSC-EV treatment interferes with CKD development and ameliorates cardiomyopathy in PNx mice.

Activation of Wnt/ß-catenin signaling is critical for the tumorigenesis of choroid plexus.

BACKGROUND: Choroid plexus (ChP) is the secretory epithelial structure located in brain ventricles. Choroid plexus tumors (CPTs) are rare neoplasms predominantly occurring in young patients with intensified malignancy in children. CPT treatment is hindered by insufficient knowledge of the tumor pathology and limited availability of valid models. METHODS: Genomic and transcriptomic data from CPT patients were analyzed to identify the putative pathological pathway. Cellular and molecular techniques were employed to validate bioinformatic results in CPT patient samples. Pharmacologic inhibition of Wnt/ß-catenin signaling was assessed in CPT cells. Cell-based assays of ChP cell lines were performed following CRISPR-Cas9-derived knockout and over-expression of Wnt/ß-catenin pathway genes. 3D CPT model was generated through CRISPR-Cas9-derived knockout of APC. RESULTS: We discovered that Wnt/ß-catenin signaling is activated in human CPTs, likely as a consequence of large-scale chromosomal instability events of the CPT genomes. We demonstrated that CPT-derived cells depend on autocrine Wnt/ß-catenin signaling for survival. Constitutive Wnt/ß-catenin pathway activation, either through knock-out of the negative regulator APC or overexpression of the ligand WNT3A, induced tumorigenic properties in ChP 2D in vitro models. Increased activation of Wnt/ß-catenin pathway in ChP organoids, through treatment with a potent GSK3ß inhibitor, reduced the differentiation of mature ChP epithelia cells. Remarkably, the depletion of APC was sufficient to induce the oncogenic transformation of ChP organoids. CONCLUSIONS: Our research identifies Wnt/ß-catenin signaling as a critical driver of CPT tumorigenesis and provides the first 3D in vitro model for future pathological and therapeutic studies of CPT.

Morphological and mitochondrial changes in murine choroid plexus epithelial cells during healthy aging.

BACKGROUND: Choroid plexuses (ChPs) are intraventricular structures mainly composed by specialized epithelial cells interconnected by tight junctions that establish the blood-cerebrospinal fluid (CSF) barrier. ChPs are essential to produce CSF and transport solutes from and into the brain. Deterioration of ChP function and morphology has been correlated to worsening of neurodegenerative disorders. We here map morpho-functional changes in the ChP epithelial cells during healthy aging, starting from young adult to 2-years old mice. METHODS: We used a multi-tiered approach, including transmission electron microscopy (TEM), immunohistochemistry, RT-qPCR, Western Blot and 2-photon microscopy (2-PM) at multiple timepoints ranging from young adult to 2-years old mice. RESULTS: We identified distinct morpho-functional modifications in epithelial cells of ChP starting from 8 to 12 months of age, which mostly remained stable up to 2 years. These changes include flattening of the epithelium, reduction of microvilli length and an augmentation of interrupted tight junctions. We also found a decrease in mitochondria density together with elongation of mitochondria in older mice. Morphological mitochondrial rearrangements were accompanied by increased superoxide levels, decreased membrane potential and decreased mitochondrial motility in aged mice. Interestingly, most of the age-related changes were not accompanied by modification of protein and/or gene expression levels and aged mitochondria effectively responded to acute pharmacological stressful stimuli. CONCLUSIONS: Our study suggests a long-term progression of multiple morpho-functional features of the mouse choroid plexus epithelium during adulthood followed by structural remodeling during the aging process. These findings can lead to a better understanding on how functional and morphological rearrangements of ChP are correlated during aging.

Choroid plexuses carry nodal-like cilia that undergo axoneme regression from early adult stage.

Choroid plexuses (ChPs) produce cerebrospinal fluid and sense non-cell-autonomous stimuli to control the homeostasis of the central nervous system. They are mainly composed of epithelial multiciliated cells, whose development and function are still controversial. We have thus characterized the stepwise order of mammalian ChP epithelia cilia formation using a combination of super-resolution-microscopy approaches and mouse genetics. We show that ChP ciliated cells are built embryonically on a treadmill of spatiotemporally regulated events, starting with atypical centriole amplification and ending with the construction of nodal-like 9+0 cilia, characterized by both primary and motile features. ChP cilia undergo axoneme resorption at early postnatal stages through a microtubule destabilization process controlled by the microtubule-severing enzyme spastin and mitigated by polyglutamylation levels. Notably, this phenotype is preserved in humans, suggesting a conserved ciliary resorption mechanism in mammals.

Increased Placental Anti-Oxidant Response in Asymptomatic and Symptomatic COVID-19 Third-Trimester Pregnancies.

Despite Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) -induced Oxidative Stress (OxS) being well documented in different organs, the molecular pathways underlying placental OxS in late-pregnancy women with SARS-CoV-2 infection are poorly understood. Herein, we performed an observational study to determine whether placentae of women testing positive for SARS-CoV-2 during the third trimester of pregnancy showed redox-related alterations involving Catalase (CAT) and Superoxide Dismutase (SOD) antioxidant enzymes as well as placenta morphological anomalies relative to a cohort of healthy pregnant women. Next, we evaluated if placental redox-related alterations and mitochondria pathological changes were correlated with the presence of maternal symptoms. We observed ultrastructural alterations of placental mitochondria accompanied by increased levels of oxidative stress markers Thiobarbituric Acid Reactive Substances (TBARS) and Hypoxia Inducible Factor-1 α (HIF-1α) in SARS-CoV-2 women during the third trimester of pregnancy. Importantly, we found an increase in placental CAT and SOD antioxidant enzymes accompanied by physiological neonatal outcomes. Our findings strongly suggest a placenta-mediated OxS inhibition in response to SARS-CoV-2 infection, thus contrasting the cytotoxic profile caused by Coronavirus Disease 2019 (COVID-19).

Interplay of BDNF and GDNF in the Mature Spinal Somatosensory System and Its Potential Therapeutic Relevance.

The growth factors BDNF and GDNF are gaining more and more attention as modulators of synaptic transmission in the mature central nervous system (CNS). The two molecules undergo a regulated secretion in neurons and may be anterogradely transported to terminals where they can positively or negatively modulate fast synaptic transmission. There is today a wide consensus on the role of BDNF as a pro-nociceptive modulator, as the neurotrophin has an important part in the initiation and maintenance of inflammatory, chronic, and/or neuropathic pain at the peripheral and central level. At the spinal level, BDNF intervenes in the regulation of chloride equilibrium potential, decreases the excitatory synaptic drive to inhibitory neurons, with complex changes in GABAergic/glycinergic synaptic transmission, and increases excitatory transmission in the superficial dorsal horn. Differently from BDNF, the role of GDNF still remains to be unraveled in full. This review resumes the current literature on the interplay between BDNF and GDNF in the regulation of nociceptive neurotransmission in the superficial dorsal horn of the spinal cord. We will first discuss the circuitries involved in such a regulation, as well as the reciprocal interactions between the two factors in nociceptive pathways. The development of small molecules specifically targeting BDNF, GDNF and/or downstream effectors is opening new perspectives for investigating these neurotrophic factors as modulators of nociceptive transmission and chronic pain. Therefore, we will finally consider the molecules of (potential) pharmacological relevance for tackling normal and pathological pain.

2D vs 3D morphological analysis of dorsal root ganglia in health and painful neuropathy.

Dorsal root ganglia (DRGs) are clusters of sensory neurons that transmit the sensory information from the periphery to the central nervous system, and satellite glial cells (SGCs), their supporting trophic cells. Sensory neurons are pseudounipolar neurons with a heterogeneous neurochemistry reflecting their functional features. DRGs, not protected by the blood brain barrier, are vulnerable to stress and damage of different origin (i.e., toxic, mechanical, metabolic, genetic) that can involve sensory neurons, SGCs or, considering their intimate intercommunication, both cell populations. DRG damage, primary or secondary to nerve damage, produces a sensory peripheral neuropathy, characterized by neurophysiological abnormalities, numbness, paraesthesia and dysesthesia, tingling and burning sensations and neuropathic pain. DRG stress can be morphologically detected by light and electron microscope analysis with alterations in cell size (swelling/atrophy) and in different sub-cellular compartments (i.e., mitochondria, endoplasmic reticulum, and nucleus) of neurons and/or SGCs. In addition, neurochemical changes can be used to portray abnormalities of neurons and SGC. Conventional immunostaining, i.e., immunohistochemical detection of specific molecules in tissue slices can be employed to detect, localize and quantify particular markers of damage in neurons (i.e., nuclear expression ATF3) or SGCs (i.e., increased expression of GFAP), markers of apoptosis (i.e., caspases), markers of mitochondrial suffering and oxidative stress (i.e., 8-OHdG), markers of tissue inflammation (i.e., CD68 for macrophage infiltration), etc. However classical (2D) methods of immunostaining disrupt the overall organization of the DRG, thus resulting in the loss of some crucial information. Whole-mount (3D) methods have been recently developed to investigate DRG morphology and neurochemistry without tissue slicing, giving the opportunity to study the intimate relationship between SGCs and sensory neurons in health and disease. Here, we aim to compare classical (2D) vs whole-mount (3D) approaches to highlight "pros" and "cons" of the two methodologies when analysing neuropathy-induced alterations in DRGs.

Increased activity and altered subcellular distribution of lysosomal enzymes determine neuronal vulnerability in Niemann-Pick type C1-deficient mice.

Niemann-Pick disease type C (NPC), caused by mutations in the Npc1 or Npc2 genes, is a progressive neurodegenerative disorder characterized by intracellular accumulation/redistribution of cholesterol in a number of tissues including the brain. This is accompanied by a severe loss of neurons in selected brain regions. In this study, we evaluated the role of lysosomal enzymes, cathepsins B and D, in determining neuronal vulnerability in NPC1-deficient (Npc1(-/-)) mouse brains. Our results showed that Npc1(-/-) mice exhibit an age-dependent degeneration of neurons in the cerebellum but not in the hippocampus. The cellular level/expression and activity of cathepsins B and D are increased more predominantly in the cerebellum than in the hippocampus of Npc1(-/-) mice. In addition, the cytosolic levels of cathepsins, cytochrome c, and Bax2 are higher in the cerebellum than in the hippocampus of Npc1(-/-) mice, suggesting a role for these enzymes in the degeneration of neurons. This suggestion is supported by our observation that degeneration of cultured cortical neurons treated with U18666A, which induces an NPC1-like phenotype at the cellular level, can be attenuated by inhibition of cathepsin B or D enzyme activity. These results suggest that the increased level/activity and altered subcellular distribution of cathepsins may be associated with the underlying cause of neuronal vulnerability in Npc1(-/-) brains. Therefore, their inhibitors may have therapeutic potential in attenuating NPC pathology.

BDNF as a pain modulator.

At least some neurotrophins may be powerful modulators of synapses, thereby influencing short- and long-term synaptic efficiency. BDNF acts at central synapses in pain pathways both at spinal and supraspinal levels. Neuronal synthesis, subcellular storage/co-storage and release of BDNF at these synapses have been characterized on anatomical and physiological grounds, in parallel with trkB (the high affinity BDNF receptor) distribution. Histological and functional evidence has been provided, mainly from studies on acute slices and intact animals, that BDNF modulates fast excitatory (glutamatergic) and inhibitory (GABAergic/glycinergic) signals, as well as slow peptidergic neurotrasmission in spinal cord. Recent studies have unraveled some of the neuronal circuitries and mechanisms involved, highlighting the key role of synaptic glomeruli in lamina II as the main sites for such a modulation.

Autophagy regulates the post-translational cleavage of BCL-2 and promotes neuronal survival.

B-cell lymphoma 2 protein (BCL-2) is one of the more widely investigated anti-apoptotic protein in mammals, and its levels are critical for protecting from programmed cell death. We report here that the cellular content of BCL-2 is regulated at post-translational level along the autophagy/lysosome pathways in organotypic cultures of post-natal mouse cerebellar cortex. Specifically this mechanism appears to be effective in the cerebellar granule cells (CGCs) that are known to undergo massive programmed cell death (apoptosis) during post-natal maturation. By the use of specific agonists/antagonist of calcium channels at the endoplasmic reticulum it was possible to understand the pivotal role of calcium release from intracellular stores in CGC neuroprotection. The more general significance of these findings is supported by a very recent study Niemann-Pick transgenic mice.