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While our understanding of the nanoscale architecture of anterograde synaptic transmission is rapidly expanding, the qualitative and quantitative molecular principles underlying distinct mechanisms of retrograde synaptic communication remain elusive. We show that a particular form of tonic cannabinoid signaling is essential for setting target cell-dependent synaptic variability. It does not require the activity of the two major endocannabinoid-producing enzymes. Instead, by developing a workflow for physiological, anatomical, and molecular measurements at the same unitary synapse, we demonstrate that the nanoscale stoichiometric ratio of type 1 cannabinoid receptors (CB1Rs) to the release machinery is sufficient to predict synapse-specific release probability. Accordingly, selective decrease of extrasynaptic CB1Rs does not affect synaptic transmission, whereas in vivo exposure to the phytocannabinoid Δ9-tetrahydrocannabinol disrupts the intrasynaptic nanoscale stoichiometry and reduces synaptic variability. These findings imply that synapses leverage the nanoscale stoichiometry of presynaptic receptor coupling to the release machinery to establish synaptic strength in a target cell-dependent manner.
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Receptor CB1 de Canabinoide , Transdução de Sinais , Sinapses , Transmissão Sináptica , Animais , Transmissão Sináptica/efeitos dos fármacos , Receptor CB1 de Canabinoide/metabolismo , Sinapses/metabolismo , Terminações Pré-Sinápticas/metabolismo , Camundongos , Endocanabinoides/metabolismo , Endocanabinoides/farmacologia , Dronabinol/farmacologiaRESUMO
The increasing prevalence of cannabis use during pregnancy has raised significant medical concerns, primarily related to the presence of Δ9-tetrahydrocannabinol (THC), which readily crosses the placenta and impacts fetal brain development. Previous research has identified midbrain dopaminergic neuronal alterations related to maternal THC consumption. However, the enduring consequences that prenatal cannabis exposure (PCE) has on striatum-based processing during voluntary reward pursuit have not been specifically determined. Here, we characterize PCE rats during food (palatable pellets) or opioid (remifentanyl)-maintained reward seeking. We find that the supra motivational phenotype of PCE rats is independent of value-based processing and is instead related to augmented reinforcing efficiency of opioid rewards. Our findings reveal that in utero THC exposure leads to increased cue-evoked dopamine release responses and an overrepresentation of cue-aligned, effort-driven striatal patterns of encoding. Recapitulating findings in humans, drug-related neurobiological adaptations of PCE were more pronounced in males, who similarly showed increased vulnerability for relapse. Collectively, these findings indicate that prenatal THC exposure in male rats engenders a pronounced neurodevelopmental susceptibility to addiction-like disorders later in life.
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Recreational use of synthetic cannabinoid agonists (i.e., "Spice" compounds) that target the Cannabinoid Type 1 receptor (CB 1 ) can cause respiratory depression in humans. However, Δ 9 -tetrahydrocannabinol (THC), the major psychoactive phytocannabinoid in cannabis, is not traditionally thought to interact with CNS control of respiration, based largely upon sparse labeling of CB1 receptors in the medulla and few reports of clinically significant respiratory depression following cannabis overdose. The respiratory effects of CB 1 agonists have rarely been studied in vivo , suggesting that additional inquiry is required to reconcile the conflict between conventional wisdom and human data. Here we used whole body plethysmography to examine the respiratory effects of the synthetic high efficacy CB 1 agonist CP55,940, and the low efficacy CB 1 agonist Δ 9 -tetrahydrocannabinol in male and female mice. CP55,940 and THC, administered systemically, both robustly suppressed minute ventilation. Both cannabinoids also produced sizable reductions in tidal volume, decreasing both peak inspiratory and expiratory flow - measures of respiratory effort. Similarly, both drugs reduced respiratory frequency, decreasing both inspiratory and expiratory time while markedly increasing expiratory pause, and to a lesser extent, inspiratory pause. Respiratory suppressive effects occurred at lower doses in females than in males, and at many of the same doses shown to produce cardinal behavioral signs of CB 1 activation. We next used RNAscope in situ hybridization to localize CB 1 mRNA to glutamatergic neurons in the medullary pre-Bötzinger Complex, a critical nucleus in controlling respiration. Our results show that, contrary to previous conventional wisdom, CB 1 mRNA is expressed in glutamatergic neurons in a brain region essential for breathing and CB 1 agonists can cause significant respiratory depression.
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Myelin protein zero (MPZ/P0) is a major structural protein of peripheral nerve myelin. Disease-associated variants in the MPZ gene cause a wide phenotypic spectrum of inherited peripheral neuropathies. Previous nerve biopsy studies showed evidence for subtype-specific morphological features. Here, we aimed at enhancing the understanding of these subtype-specific features and pathophysiological aspects of MPZ neuropathies. We examined archival material from two Central European centers and systematically determined genetic, clinical, and neuropathological features of 21 patients with MPZ mutations compared to 16 controls. Cases were grouped based on nerve conduction data into congenital hypomyelinating neuropathy (CHN; n = 2), demyelinating Charcot-Marie-Tooth (CMT type 1; n = 11), intermediate (CMTi; n = 3), and axonal CMT (type 2; n = 5). Six cases had combined muscle and nerve biopsies and one underwent autopsy. We detected four MPZ gene variants not previously described in patients with neuropathy. Light and electron microscopy of nerve biopsies confirmed fewer myelinated fibers, more onion bulbs and reduced regeneration in demyelinating CMT1 compared to CMT2/CMTi. In addition, we observed significantly more denervated Schwann cells, more collagen pockets, fewer unmyelinated axons per Schwann cell unit and a higher density of Schwann cell nuclei in CMT1 compared to CMT2/CMTi. CHN was characterized by basal lamina onion bulb formation, a further increase in Schwann cell density and hypomyelination. Most late onset axonal neuropathy patients showed microangiopathy. In the autopsy case, we observed prominent neuromatous hyperinnervation of the spinal meninges. In four of the six muscle biopsies, we found marked structural mitochondrial abnormalities. These results show that MPZ alterations not only affect myelinated nerve fibers, leading to either primarily demyelinating or axonal changes, but also affect non-myelinated nerve fibers. The autopsy case offers insight into spinal nerve root pathology in MPZ neuropathy. Finally, our data suggest a peculiar association of MPZ mutations with mitochondrial alterations in muscle.
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Doença de Charcot-Marie-Tooth , Proteína P0 da Mielina , Humanos , Proteína P0 da Mielina/genética , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/patologia , Mutação/genética , Proteínas/genética , BiópsiaRESUMO
Membrane-shaping proteins characterized by reticulon homology domains play an important part in the dynamic remodelling of the endoplasmic reticulum (ER). An example of such a protein is FAM134B, which can bind LC3 proteins and mediate the degradation of ER sheets through selective autophagy (ER-phagy)1. Mutations in FAM134B result in a neurodegenerative disorder in humans that mainly affects sensory and autonomic neurons2. Here we report that ARL6IP1, another ER-shaping protein that contains a reticulon homology domain and is associated with sensory loss3, interacts with FAM134B and participates in the formation of heteromeric multi-protein clusters required for ER-phagy. Moreover, ubiquitination of ARL6IP1 promotes this process. Accordingly, disruption of Arl6ip1 in mice causes an expansion of ER sheets in sensory neurons that degenerate over time. Primary cells obtained from Arl6ip1-deficient mice or from patients display incomplete budding of ER membranes and severe impairment of ER-phagy flux. Therefore, we propose that the clustering of ubiquitinated ER-shaping proteins facilitates the dynamic remodelling of the ER during ER-phagy and is important for neuronal maintenance.
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Autofagia , Estresse do Retículo Endoplasmático , Retículo Endoplasmático , Proteínas Ubiquitinadas , Ubiquitinação , Animais , Humanos , Camundongos , Autofagia/genética , Retículo Endoplasmático/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Ubiquitinadas/metabolismo , Células Receptoras Sensoriais/metabolismo , Células Receptoras Sensoriais/patologia , Membranas Intracelulares/metabolismoRESUMO
Proteins of the secretory pathway undergo glycosylation in the endoplasmic reticulum (ER) and the Golgi apparatus. Altered protein glycosylation can manifest in serious, sometimes fatal malfunctions. We recently showed that mutations in GDP-mannose pyrophosphorylase A (GMPPA) can cause a syndrome characterized by alacrima, achalasia, mental retardation, and myopathic alterations (AAMR syndrome). GMPPA acts as a feedback inhibitor of GDP-mannose pyrophosphorylase B (GMPPB), which provides GDP-mannose as a substrate for protein glycosylation. Loss of GMPPA thus enhances the incorporation of mannose into glycochains of various proteins, including α-dystroglycan (α-DG), a protein that links the extracellular matrix with the cytoskeleton. Here, we further characterized the consequences of loss of GMPPA for the secretory pathway. This includes a fragmentation of the Golgi apparatus, which comes along with a regulation of the abundance of several ER- and Golgi-resident proteins. We further show that the activity of the Golgi-associated endoprotease furin is reduced. Moreover, the fraction of α-DG, which is retained in the ER, is increased. Notably, WT cells cultured at a high mannose concentration display similar changes with increased retention of α-DG, altered structure of the Golgi apparatus, and a decrease in furin activity. In summary, our data underline the importance of a balanced mannose homeostasis for the secretory pathway.
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BACKGROUND AND PURPOSE: Ehlers-Danlos syndromes are hereditary disorders of connective tissue that are characterized by joint hypermobility, skin hyperextensibility and tissue fragility. The most common subtype is the hypermobile type. In addition to symptoms of small fibre neuropathy (SFN) due to damage to the small peripheral nerve fibres, with degeneration of the distal nerve endings, autonomic disorders such as postural tachycardia syndrome (PoTS) are frequently reported features in patients with hypermobile Ehlers-Danlos syndrome (hEDS). To date, the underlying pathophysiological mechanisms are still not completely understood. STUDY PURPOSE: To better understand pathophysiological mechanisms of small fiber neuropathy and autonomic neuropathy in hypermobile Ehlers-Danlos Syndromes. METHODS: We prospectively investigated 31 patients with hEDS compared to 31 healthy controls by using skin biopsy, quantitative sensory testing, tilt-table testing, the painDetect, Small Fibre Neuropathy Screening List and the COMPASS-31 (Composite Autonomic Symptom Score 31) questionnaire. RESULTS: Nineteen (61%) patients with hEDS were diagnosed with SFN, and 10 (32%) fulfilled the criteria for PoTS. Patients with hEDS had significantly higher heart rates than controls. According to quantitative sensory testing, these patients had generalized thermal and tactile hypesthesia. Skin biopsy revealed significantly reduced intraepithelial nerve fibre density proximally (thigh) and distally (lower leg) in patients compared to controls. This was consistent with various complaints of pain and sensory disturbances in both the proximal and distal body regions. CONCLUSION: These results confirm histologically proven SFN as a common feature in patients with hEDS, revealing a generalized distribution of nerve fibre loss. Regarding the frequently reported autonomic and neuropathic dysfunctions, the findings support SFN as an important, but not the only, underlying pathomechanism.
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Síndrome de Ehlers-Danlos , Neuropatia de Pequenas Fibras , Humanos , Neuropatia de Pequenas Fibras/etiologia , Síndrome de Ehlers-Danlos/complicações , Síndrome de Ehlers-Danlos/diagnóstico , Síndrome de Ehlers-Danlos/patologia , Pele/patologia , BiópsiaRESUMO
Single-molecule localization microscopy (SMLM) opened new possibilities to study the spatial arrangement of molecular distribution and disease-associated redistribution at a previously unprecedented resolution that was not achievable with optical microscopy approaches. Recent discoveries based on SMLM techniques uncovered specific nanoscale organizational principles of signaling proteins in several biological systems including the chemical synapses in the brain. Emerging data suggest that the spatial arrangement of the molecular players of the endocannabinoid system is also precisely regulated at the nanoscale level in synapses and in other neuronal and glial subcellular compartments. The precise nanoscale distribution pattern is likely to be important to subserve several specific signaling functions of this important messenger system in a cell-type- and subcellular domain-specific manner.STochastic Optical Reconstruction Microscopy (STORM) is an especially suitable SMLM modality for cell-type-specific nanoscale molecular imaging due to its compatibility with traditional diffraction-limited microscopy approaches and classical staining methods. Here, we describe a detailed protocol for STORM imaging in mouse brain tissue samples with a focus on the CB1 cannabinoid receptor, one of the most abundant synaptic receptors in the brain. We also summarize important conceptual and methodical details that are essential for the valid interpretation of single-molecule localization microscopy data.
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Endocanabinoides , Microscopia , Animais , Endocanabinoides/metabolismo , Camundongos , Microscopia/métodos , Receptores de Canabinoides , Receptores de Neurotransmissores , Sinapses/metabolismoRESUMO
Here, we studied the neuroinflammation- and ischemia-related glial markers chitotriosidase 1 (CHIT1) and chitinase-3-like protein 1 (CHI3L1, alias YKL-40) in the human striate cortex and cerebellum at different time points after global hypoxic-ischemic brain injury (HIBI). Both regions differ considerably in their glial cell population but are supplied by the posterior circulation. CHIT1 and CHI3L1 expression was compared to changes in microglial (IBA1, CD68), astrocytic (GFAP, S100ß), and neuronal markers (H&E, neurofilament heavy chain, NfH; calretinin, CALR) using immunohistochemistry and multiple-label immunofluorescence. Initial striatal cortical and cerebellar Purkinje cell damage, detectable already 1/2 d after HIBI, led to delayed neuronal death, whereas loss of cerebellar NfH-positive stellate and CALR-positive granule cells was variable. During the first week post-HIBI, a transient reduction of IBA1-positive microglia was observed in both regions, and fragmented/clasmatodendritic cerebellar Bergmann glia appeared. In long-term survivors, both brain regions displayed high densities of activated IBA1-positive cells and CD68-positive macrophages, which showed CHIT1 co-localization in the striate cortex. Furthermore, enlarged GFAP- and S100ß-positive astroglia emerged in both regions around 9-10 d post-HIBI, i.e., along with clearance of dead neurons from the neuropil, although GFAP-/S100ß-positive gemistocytic astrocytes that co-expressed CHI3L1 were found only in the striate cortex. Thus, only GFAP-/S100ß-positive astrocytes in the striate cortex, but not cerebellar Bergmann glia, differentiated into CHI3L1-positive gemistocytes. CHIT1 was co-expressed almost entirely in macrophages in the striate cortex and not cerebellum of long-term survivors, thereby indicating that CHIT1 and CHI3L1 could be valuable biomarkers for monitoring the outcome of global HIBI.
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Quitinases , Humanos , Córtex Visual Primário , Neuroglia , Hipóxia , IsquemiaRESUMO
Microglia, the resident immune cells of the brain, play important roles during development. Although bi-directional communication between microglia and neuronal progenitors or immature neurons has been demonstrated, the main sites of interaction and the underlying mechanisms remain elusive. By using advanced methods, here we provide evidence that microglial processes form specialized contacts with the cell bodies of developing neurons throughout embryonic, early postnatal, and adult neurogenesis. These early developmental contacts are highly reminiscent of somatic purinergic junctions that are instrumental for microglia-neuron communication in the adult brain. The formation and maintenance of these junctions is regulated by functional microglial P2Y12 receptors, and deletion of P2Y12Rs disturbs proliferation of neuronal precursors and leads to aberrant cortical cytoarchitecture during development and in adulthood. We propose that early developmental formation of somatic purinergic junctions represents an important interface for microglia to monitor the status of immature neurons and control neurodevelopment.
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Microglia , Neurogênese , Adulto , Encéfalo , Humanos , Microglia/fisiologia , Neurônios/fisiologiaRESUMO
OBJECTIVE: Autonomic small fibre neuropathy is described in patients with autoimmune autonomic neuropathy (AAN). Few data are available on somatosensory function and skin biopsies in AAN. METHODS: Retrospective analysis of 17 patients (51.2 ± 6.8 years, n = 7 males) with AAN, including autoantibodies, quantitative sensory testing (QST, n = 13) and intraepithelial nerve fibre density (IENFD) in skin biopsy (n = 16). QST was performed according to the DFNS protocol over hands and feet dorsum. QST data were compared to healthy controls. Comparison of antibody-positive and antibody-negative cases. RESULTS: 70.6% of patients were antibody positive. 82.4% described at least one episode with sensory symptoms. Skin biopsies revealed reduced IENFD in 58.8% of patients, whereas neuropathic pain was only present in 41.2%. QST showed a nonregional increase for nonpainful thermal and mechanical detection rather than for mechanical pain thresholds. Compared to healthy controls, sensory loss for cold and warm detection thresholds and for the thermal sensory limen-the temperature difference between alternating warm and cold stimuli-was found on hands and feet (all p < 0.05). For nonpainful mechanical stimuli, the vibration detection threshold on the hand was increased (p < 0.05). Of all pain thresholds, only the mechanical pain threshold was elevated for pinprick stimuli to the feet (p < 0.05). INTERPRETATION: Findings are consistent with a sensory small fibre more than large fibre neuropathy in AAN. Sensory loss was comparably distributed across hands and feet, indicating that nerve fibre dysfunction was rather generalized. Serostatus was not a significant predictor of the small fibre deficit present in AAN.
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Neuralgia , Neuropatia de Pequenas Fibras , Masculino , Humanos , Neuropatia de Pequenas Fibras/complicações , Neuropatia de Pequenas Fibras/diagnóstico , Estudos Retrospectivos , Limiar da Dor/fisiologia , Fibras Nervosas/patologia , Neuralgia/etiologia , Neuralgia/patologiaRESUMO
Eliciting regulated cell death, like necroptosis, is a potential cancer treatment. However, pathways eliciting necroptosis are poorly understood. It has been reported that prolonged activation of acid-sensing ion channel 1a (ASIC1a) induces necroptosis in mouse neurons. Glioblastoma stem cells (GSCs) also express functional ASIC1a, but whether prolonged activation of ASIC1a induces necroptosis in GSCs is unknown. Here we used a tumorsphere formation assay to show that slight acidosis (pH 6.6) induces necrotic cell death in a manner that was sensitive to the necroptosis inhibitor Nec-1 and to the ASIC1a antagonist PcTx1. In addition, genetic knockout of ASIC1a rendered GSCs resistant to acid-induced reduction in tumorsphere formation, while the ASIC1 agonist MitTx1 reduced tumorsphere formation also at neutral pH. Finally, a 20 amino acid fragment of the ASIC1 C-terminus, thought to interact with the necroptosis kinase RIPK1, was sufficient to reduce the formation of tumorspheres. Meanwhile, the genetic knockout of MLKL, the executive protein in the necroptosis cascade, did not prevent a reduction in tumor sphere formation, suggesting that ASIC1a induced an alternative cell death pathway. These findings demonstrate that ASIC1a is a death receptor on GSCs that induces cell death during prolonged acidosis. We propose that this pathway shapes the evolution of a tumor in its acidic microenvironment and that pharmacological activation of ASIC1a might be a potential new strategy in tumor therapy.
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Acidose , Glioblastoma , Canais Iônicos Sensíveis a Ácido/genética , Canais Iônicos Sensíveis a Ácido/metabolismo , Acidose/metabolismo , Animais , Glioblastoma/genética , Glioblastoma/metabolismo , Camundongos , Neurônios/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Células-Tronco/metabolismo , Microambiente TumoralRESUMO
Numerous intervention strategies have been developed to promote functional tissue repair following experimental spinal cord injury (SCI), including the bridging of lesion-induced cystic cavities with bioengineered scaffolds. Integration between such implanted scaffolds and the lesioned host spinal cord is critical for supporting regenerative growth, but only moderate-to-low degrees of success have been reported. Light and electron microscopy were employed to better characterise the fibroadhesive scarring process taking place after implantation of a longitudinally microstructured type-I collagen scaffold into unilateral mid-cervical resection injuries of the adult rat spinal cord. At long survival times (10 weeks post-surgery), sheets of tightly packed cells (of uniform morphology) could be seen lining the inner surface of the repaired dura mater of lesion-only control animals, as well as forming a barrier along the implant-host interface of the scaffold-implanted animals. The highly uniform ultrastructural features of these scarring cells and their anatomical continuity with the local, reactive spinal nerve roots strongly suggest their identity to be perineurial-like cells. This novel aspect of the cellular composition of reactive spinal cord tissue highlights the increasingly complex nature of fibroadhesive scarring involved in traumatic injury, and particularly in response to the implantation of bioengineered collagen scaffolds.
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Colágeno Tipo I , Traumatismos da Medula Espinal , Animais , Cicatriz/patologia , Colágeno/química , Regeneração Nervosa/fisiologia , Ratos , Medula Espinal/patologia , Traumatismos da Medula Espinal/patologia , Alicerces Teciduais/químicaRESUMO
ClC-3, ClC-4, and ClC-5 are electrogenic chloride/proton exchangers that can be found in endosomal compartments of mammalian cells. Although the association with genetic diseases and the severe phenotype of knock-out animals illustrate their physiological importance, the cellular functions of these proteins have remained insufficiently understood. We here study the role of two Clcn3 splice variants, ClC-3b and ClC-3c, in granular exocytosis and catecholamine accumulation of adrenal chromaffin cells using a combination of high-resolution capacitance measurements, amperometry, protein expression/gene knock out/down, rescue experiments, and confocal microscopy. We demonstrate that ClC-3c resides in immature as well as in mature secretory granules, where it regulates catecholamine accumulation and contributes to the establishment of the readily releasable pool of secretory vesicles. The lysosomal splice variant ClC-3b contributes to vesicle priming only with low efficiency and leaves the vesicular catecholamine content unaltered. The related Cl-/H+ antiporter ClC-5 undergoes age-dependent downregulation in wild-type conditions. Its upregulation in Clcn3-/- cells partially rescues the exocytotic mutant defect. Our study demonstrates how different CLC transporters with similar transport functions, but distinct localizations can contribute to vesicle functions in the regulated secretory pathway of granule secretion in chromaffin cells.SIGNIFICANCE STATEMENT Cl-/H+ exchangers are expressed along the endosomal/lysosomal system of mammalian cells; however, their exact subcellular functions have remained insufficiently understood. We used chromaffin cells, a system extensively used to understand presynaptic mechanisms of synaptic transmission, to define the role of CLC exchangers in neurosecretion. Disruption of ClC-3 impairs catecholamine accumulation and secretory vesicle priming. There are multiple ClC-3 splice variants, and only expression of one, ClC-3c, in double Cl-/H+ exchanger-deficient cells fully rescues the WT phenotype. Another splice variant, ClC-3b, is present in lysosomes and is not necessary for catecholamine secretion. The distinct functions of ClC-3c and ClC-3b illustrate the impact of expressing multiple CLC transporters with similar transport functions and separate localizations in different endosomal compartments.
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Células Cromafins , Prótons , Animais , Catecolaminas/metabolismo , Cloretos/metabolismo , Células Cromafins/metabolismo , Exocitose/fisiologia , Mamíferos , Camundongos , Camundongos Knockout , Vesículas Secretórias/metabolismoRESUMO
It is always a challenge to acquire a clear picture of the pathological processes and changes in any disease. For this purpose, it is advantageous to directly examine the affected organ. Nerve biopsy has been a method of choice for decades to classify peripheral neuropathies and to find clues to uncover their etiology. The histologic examination of the peripheral nerve provides information on axonal or myelin pathology as well as on the surrounding connective tissue and vascularization of the nerve. Minimal requirements of the workup include paraffin histology as well as resin semithin section histology. Cryostat sections, teased fiber preparations and electron microscopy are potentially useful in a subset of cases. Here we describe our standard procedures for the workup of the tissue sample and provide examples of diagnostically relevant findings.
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Doenças do Sistema Nervoso Periférico , Axônios/patologia , Biópsia/métodos , Humanos , Bainha de Mielina/patologia , Nervos Periféricos/patologia , Doenças do Sistema Nervoso Periférico/diagnóstico , Doenças do Sistema Nervoso Periférico/patologia , Nervo Sural/patologiaRESUMO
Immunolabeling and autoradiography have traditionally been applied as the methods-of-choice to visualize and collect molecular information about physiological and pathological processes. Here, we introduce PharmacoSTORM super-resolution imaging that combines the complementary advantages of these approaches and enables cell-type- and compartment-specific nanoscale molecular measurements. We exploited rational chemical design for fluorophore-tagged high-affinity receptor ligands and an enzyme inhibitor; and demonstrated broad PharmacoSTORM applicability for three protein classes and for cariprazine, a clinically approved antipsychotic and antidepressant drug. Because the neurobiological substrate of cariprazine has remained elusive, we took advantage of PharmacoSTORM to provide in vivo evidence that cariprazine predominantly binds to D3 dopamine receptors on Islands of Calleja granule cell axons but avoids dopaminergic terminals. These findings show that PharmacoSTORM helps to quantify drug-target interaction sites at the nanoscale level in a cell-type- and subcellular context-dependent manner and within complex tissue preparations. Moreover, the results highlight the underappreciated neuropsychiatric significance of the Islands of Calleja in the ventral forebrain.
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Ínsulas Olfatórias/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Piperazinas/farmacologia , Receptores de Dopamina D2/metabolismo , Receptores de Dopamina D3/metabolismoRESUMO
Mutations in RNA binding proteins (RBPs) and in genes regulating autophagy are frequent causes of familial amyotrophic lateral sclerosis (fALS). The P56S mutation in vesicle-associated membrane protein-associated protein B (VAPB) leads to fALS (ALS8) and spinal muscular atrophy (SMA). While VAPB is primarily involved in the unfolded protein response (UPR), vesicular trafficking and in initial steps of the autophagy pathway, the effect of mutant P56S-VAPB on autophagy regulation in connection with RBP homeostasis has not been explored yet. Examining the muscle biopsy of our index ALS8 patient of European origin revealed globular accumulations of VAPB aggregates co-localised with autophagy markers LC3 and p62 in partially atrophic and atrophic muscle fibres. In line with this skin fibroblasts obtained from the same patient showed accumulation of P56S-VAPB aggregates together with LC3 and p62. Detailed investigations of autophagic flux in cell culture models revealed that P56S-VAPB alters both initial and late steps of the autophagy pathway. Accordingly, electron microscopy complemented with live cell imaging highlighted the impaired fusion of accumulated autophagosomes with lysosomes in cells expressing P56S-VAPB. Consistent with these observations, neuropathological studies of brain and spinal cord of P56S-VAPB transgenic mice revealed signs of neurodegeneration associated with altered protein quality control and defective autophagy. Autophagy and RBP homeostasis are interdependent, as demonstrated by the cytoplasmic mis-localisation of several RBPs including pTDP-43, FUS, Matrin 3 which often sequestered with P56S-VAPB aggregates both in cell culture and in the muscle biopsy of the ALS8 patient. Further confirming the notion that aggregation of the RBPs proceeds through the stress granule (SG) pathway, we found persistent G3BP- and TIAR1-positive SGs in P56S-VAPB expressing cells as well as in the ALS8 patient muscle biopsy. We conclude that P56S-VAPB-ALS8 involves a cohesive pathomechanism of aberrant RBP homeostasis together with dysfunctional autophagy.
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Esclerose Lateral Amiotrófica/patologia , Proteínas de Ligação a RNA/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Autofagia , Homeostase , Humanos , MutaçãoRESUMO
GDP-mannose-pyrophosphorylase-B (GMPPB) facilitates the generation of GDP-mannose, a sugar donor required for glycosylation. GMPPB defects cause muscle disease due to hypoglycosylation of α-dystroglycan (α-DG). Alpha-DG is part of a protein complex, which links the extracellular matrix with the cytoskeleton, thus stabilizing myofibers. Mutations of the catalytically inactive homolog GMPPA cause alacrima, achalasia, and mental retardation syndrome (AAMR syndrome), which also involves muscle weakness. Here, we showed that Gmppa-KO mice recapitulated cognitive and motor deficits. As structural correlates, we found cortical layering defects, progressive neuron loss, and myopathic alterations. Increased GDP-mannose levels in skeletal muscle and in vitro assays identified GMPPA as an allosteric feedback inhibitor of GMPPB. Thus, its disruption enhanced mannose incorporation into glycoproteins, including α-DG in mice and humans. This increased α-DG turnover and thereby lowered α-DG abundance. In mice, dietary mannose restriction beginning after weaning corrected α-DG hyperglycosylation and abundance, normalized skeletal muscle morphology, and prevented neuron degeneration and the development of motor deficits. Cortical layering and cognitive performance, however, were not improved. We thus identified GMPPA defects as the first congenital disorder of glycosylation characterized by α-DG hyperglycosylation, to our knowledge, and we have unraveled underlying disease mechanisms and identified potential dietary treatment options.
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Distroglicanas , Guanosina Difosfato Manose , Músculo Esquelético/metabolismo , Doenças Neuromusculares , Nucleotidiltransferases/deficiência , Animais , Distroglicanas/genética , Distroglicanas/metabolismo , Glicosilação , Guanosina Difosfato Manose/genética , Guanosina Difosfato Manose/metabolismo , Humanos , Camundongos , Camundongos Knockout , Doenças Neuromusculares/dietoterapia , Doenças Neuromusculares/genética , Doenças Neuromusculares/metabolismo , Nucleotidiltransferases/metabolismoRESUMO
Galactic cosmic radiation (GCR), composed of highly energetic and fully ionized atomic nuclei, produces diverse deleterious effects on the body. In researching the neurological risks of GCR exposures, including during human spaceflight, various ground-based single-ion GCR irradiation paradigms induce differential disruptions of cellular activity and overall behavior. However, it remains less clear how irradiation comprising a mix of multiple ions, more accurately recapitulating the space GCR environment, impacts the central nervous system. We therefore examined how mixed-ion GCR irradiation (two similar 5-6 beam combinations of protons, helium, oxygen, silicon and iron ions) influenced neuronal connectivity, functional generation of activity within neural circuits and cognitive behavior in mice. In electrophysiological recordings we find that space-relevant doses of mixed-ion GCR preferentially alter hippocampal inhibitory neurotransmission and produce related disruptions in the local field potentials of hippocampal oscillations. Such underlying perturbation in hippocampal network activity correspond with perturbed learning, memory and anxiety behavior.
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Radiação Cósmica/efeitos adversos , Hipocampo/efeitos da radiação , Transmissão Sináptica/efeitos da radiação , Animais , Comportamento Animal/efeitos da radiação , Disfunção Cognitiva/etiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The molecular repertoire of the "Ca2+-signaling toolkit" supports the specific kinetic requirements of Ca2+-dependent processes in different neuronal types. A well-known example is the unique expression pattern of calcium-binding proteins, such as parvalbumin, calbindin, and calretinin. These cytosolic Ca2+-buffers control presynaptic and somatodendritic processes in a cell-type-specific manner and have been used as neurochemical markers of GABAergic interneuron types for decades. Surprisingly, to date no typifying calcium-binding proteins have been found in CB1 cannabinoid receptor/cholecystokinin (CB1/CCK)-positive interneurons that represent a large population of GABAergic cells in cortical circuits. Because CB1/CCK-positive interneurons display disparate presynaptic and somatodendritic Ca2+-transients compared with other interneurons, we tested the hypothesis that they express alternative calcium-binding proteins. By in silico data mining in mouse single-cell RNA-seq databases, we identified high expression of Necab1 and Necab2 genes encoding N-terminal EF-hand calcium-binding proteins 1 and 2, respectively, in CB1/CCK-positive interneurons. Fluorescent in situ hybridization and immunostaining revealed cell-type-specific distribution of NECAB1 and NECAB2 throughout the isocortex, hippocampal formation, and basolateral amygdala complex. Combination of patch-clamp electrophysiology, confocal, and STORM super-resolution microscopy uncovered subcellular nanoscale differences indicating functional division of labor between the two calcium-binding proteins. These findings highlight NECAB1 and NECAB2 as predominant calcium-binding proteins in CB1/CCK-positive interneurons.