RESUMEN
Muscular dystrophies (MDs) are a heterogeneous group of myopathies characterized by progressive muscle weakness leading to death from heart or respiratory failure. MDs are caused by mutations in genes involved in both the development and organization of muscle fibers. Several animal models harboring mutations in MD-associated genes have been developed so far. Together with rodents, the zebrafish is one of the most popular animal models used to reproduce MDs because of the high level of sequence homology with the human genome and its genetic manipulability. This review describes the most important zebrafish mutant models of MD and the most advanced tools used to generate and characterize all these valuable transgenic lines. Zebrafish models of MDs have been generated by introducing mutations to muscle-specific genes with different genetic techniques, such as (i) N-ethyl-N-nitrosourea (ENU) treatment, (ii) the injection of specific morpholino, (iii) tol2-based transgenesis, (iv) TALEN, (v) and CRISPR/Cas9 technology. All these models are extensively used either to study muscle development and function or understand the pathogenetic mechanisms of MDs. Several tools have also been developed to characterize these zebrafish models by checking (i) motor behavior, (ii) muscle fiber structure, (iii) oxidative stress, and (iv) mitochondrial function and dynamics. Further, living biosensor models, based on the expression of fluorescent reporter proteins under the control of muscle-specific promoters or responsive elements, have been revealed to be powerful tools to follow molecular dynamics at the level of a single muscle fiber. Thus, zebrafish models of MDs can also be a powerful tool to search for new drugs or gene therapies able to block or slow down disease progression.
Asunto(s)
Enfermedades Musculares , Distrofias Musculares , Animales , Humanos , Pez Cebra/genética , Distrofias Musculares/genética , Animales Modificados Genéticamente/genética , Fibras Musculares Esqueléticas/patologíaRESUMEN
Neuronal dendritic arborizations and dendritic spines are crucial for a normal synaptic transmission and may be critically involved in the pathophysiology of epilepsy. Alterations in dendritic morphology and spine loss mainly in hippocampal neurons have been reported both in epilepsy animal models and in human brain tissues from patients with epilepsy. However, it is still unclear whether these dendritic abnormalities relate to the cause of epilepsy or are generated by seizure recurrence. We investigated fine neuronal structures at the level of dendritic and spine organization using Golgi impregnation, and analysed synaptic networks with immunohistochemical markers of glutamatergic (vGLUT1) and GABAergic (vGAT) axon terminals in human cerebral cortices derived from epilepsy surgery. Specimens were obtained from 28 patients with different neuropathologically defined aetiologies: type Ia and type II focal cortical dysplasia, cryptogenic (no lesion) and temporal lobe epilepsy with hippocampal sclerosis. Autoptic tissues were used for comparison. Three-dimensional reconstructions of Golgi-impregnated neurons revealed severe dendritic reshaping and spine alteration in the core of the type II focal cortical dysplasia. Dysmorphic neurons showed increased dendritic complexity, reduction of dendritic spines and occasional filopodia-like protrusions emerging from the soma. Surprisingly, the intermingled normal-looking pyramidal neurons also showed severe spine loss and simplified dendritic arborization. No changes were observed outside the dysplasia (perilesional tissue) or in neocortical postsurgical tissue obtained in the other patient groups. Immunoreactivities of vGLUT1 and vGAT showed synaptic reorganization in the core of type II dysplasia characterized by the presence of abnormal perisomatic baskets around dysmorphic neurons, in particular those with filopodia-like protrusions, and changes in vGLUT1/vGAT expression. Ultrastructural data in type II dysplasia highlighted the presence of altered neuropil engulfed by glial processes. Our data indicate that the fine morphological aspect of neurons and dendritic spines are normal in epileptogenic neocortex, with the exception of type II dysplastic lesions. The findings suggest that the mechanisms leading to this severe form of cortical malformation interfere with the normal dendritic arborization and synaptic network organization. The data argue against the concept that long-lasting epilepsy and seizure recurrence per se unavoidably produce a dendritic pathology.
Asunto(s)
Corteza Cerebral/ultraestructura , Dendritas/ultraestructura , Espinas Dendríticas/ultraestructura , Epilepsia/patología , Sinapsis/ultraestructura , Adolescente , Adulto , Corteza Cerebral/metabolismo , Preescolar , Humanos , Lactante , Microscopía Electrónica , Persona de Mediana Edad , Sinapsis/metabolismo , Proteína 1 de Transporte Vesicular de Glutamato/metabolismo , Proteínas del Transporte Vesicular de Aminoácidos Inhibidores/metabolismo , Adulto JovenRESUMEN
An increased incidence in the sleep-disorder narcolepsy has been associated with the 2009-2010 pandemic of H1N1 influenza virus in China and with mass vaccination campaigns against influenza during the pandemic in Finland and Sweden. Pathogenetic mechanisms of narcolepsy have so far mainly focused on autoimmunity. We here tested an alternative working hypothesis involving a direct role of influenza virus infection in the pathogenesis of narcolepsy in susceptible subjects. We show that infection with H1N1 influenza virus in mice that lack B and T cells (Recombinant activating gene 1-deficient mice) can lead to narcoleptic-like sleep-wake fragmentation and sleep structure alterations. Interestingly, the infection targeted brainstem and hypothalamic neurons, including orexin/hypocretin-producing neurons that regulate sleep-wake stability and are affected in narcolepsy. Because changes occurred in the absence of adaptive autoimmune responses, the findings show that brain infections with H1N1 virus have the potential to cause per se narcoleptic-like sleep disruption.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A/fisiología , Narcolepsia/fisiopatología , Narcolepsia/virología , Neuronas/fisiología , Sueño , Vigilia , Animales , Antígenos Virales/inmunología , Electroencefalografía , Proteínas de Homeodominio/metabolismo , Hipotálamo/fisiopatología , Hipotálamo/virología , Inmunidad Innata , Ratones , Ratones Endogámicos C57BL , Modelos Neurológicos , Bulbo Olfatorio/fisiopatología , Bulbo Olfatorio/virología , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/fisiopatología , Infecciones por Orthomyxoviridae/virologíaRESUMEN
The infection by Trypanosoma brucei brucei (T.b.b.), a protozoan parasite, is characterized by an early-systemic stage followed by a late stage in which parasites invade the brain parenchyma in a T cell-dependent manner. Here we found that early after infection effector-memory T cells were predominant among brain T cells, whereas, during the encephalitic stage T cells acquired a tissue resident memory phenotype (TRM) and expressed PD1. Both CD4 and CD8 T cells were independently redundant for the penetration of T.b.b. and other leukocytes into the brain parenchyma. The role of lymphoid cells during the T.b.b. infection was studied by comparing T- and B-cell deficient rag1-/- and WT mice. Early after infection, parasites located in circumventricular organs, brain structures with increased vascular permeability, particularly in the median eminence (ME), paced closed to the sleep-wake regulatory arcuate nucleus of the hypothalamus (Arc). Whereas parasite levels in the ME were higher in rag1-/- than in WT mice, leukocytes were instead reduced. Rag1-/- infected mice showed increased levels of meca32 mRNA coding for a blood /hypothalamus endothelial molecule absent in the blood-brain-barrier (BBB). Both immune and metabolic transcripts were elevated in the ME/Arc of WT and rag1-/- mice early after infection, except for ifng mRNA, which levels were only increased in WT mice. Finally, using a non-invasive sleep-wake cycle assessment method we proposed a putative role of lymphocytes in mediating sleep alterations during the infection with T.b.b. Thus, the majority of T cells in the brain during the early stage of T.b.b. infection expressed an effector-memory phenotype while TRM cells developed in the late stage of infection. T cells and parasites invade the ME/Arc altering the metabolic and inflammatory responses during the early stage of infection and modulating sleep disturbances.
Asunto(s)
Enfermedades del Sistema Nervioso Central/inmunología , Enfermedades del Sistema Nervioso Central/parasitología , Subgrupos de Linfocitos T/fisiología , Trypanosoma brucei brucei , Tripanosomiasis Africana/inmunología , Tripanosomiasis Africana/patología , Animales , Encéfalo/parasitología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Memoria Inmunológica , Leucocitos , Ratones , Ratones Noqueados , SueñoRESUMEN
Memory reconsolidation is a process allowing previously consolidated memories to be updated. In order for memory reconsolidation to occur, a memory first needs to be reactivated. It has been shown recently that memory retrieval during awake/sleep phases may affect susceptibility to memory reactivation. Given the importance of hippocampal gamma frequencies in memory processes, the purpose of the present research was to study changes in gamma bands power during retrieval of instrumental appetitive memories. Local field potentials were recorded in the CA1 area of dorsal hippocampus of Sprague Dawley rats during retrieval of instrumental appetitive memory performed either during light or dark phases of the circadian cycle. Appetitive memory retrieval was performed by using a protocol of sucrose self-administration in operant chambers equipped with levers (Piva et al., 2018): rats were first trained to self-administer sucrose pellets and, after a 14-days forced abstinence stage, memory retrieval stage consisted in training context exposure. At the retrival stage performed during the light phase, a decreased low-gamma power was observed in CA1 when rats were not lever pressing compared to when they were lever pressing (actual instrumental memory retrieval). Moreover, results showed an inverse correlation between gamma power and rate of responding when retrieval was performed in the dark phase. Our findings suggest that hippocampal gamma power is differently modulated when retrieval is performed during the light phase compared to the dark phase. Further investigations should explore the role of gamma oscillations as potential markers of instrumental appetitive memory reactivation in both light and dark conditions.
Asunto(s)
Región CA1 Hipocampal/fisiología , Ritmo Gamma/fisiología , Memoria/fisiología , Sueño/fisiología , Vigilia/fisiología , Animales , Condicionamiento Operante , Masculino , Ratas , Ratas Sprague-Dawley , Sacarosa/administración & dosificaciónRESUMEN
Cullin-RING ligases (CRLs) control key cellular processes by promoting ubiquitylation of a multitude of soluble cytosolic and nuclear proteins. Subsets of CRL complexes are recruited and activated locally at cellular membranes; however, few CRL functions and substrates at these distinct cellular compartments are known. Here, we use a proteomic screen to identify proteins that are ubiquitylated at cellular membranes and found that Lunapark, an endoplasmic reticulum (ER)-shaping protein localized to ER three-way junctions, is ubiquitylated by the CRL3KLHL12 ubiquitin ligase. We demonstrate that Lunapark interacts with mechanistic target of rapamycin complex-1 (mTORC1), a central cellular regulator that coordinates growth and metabolism with environmental conditions. We show that mTORC1 binds Lunapark specifically at three-way junctions, and lysosomes, where mTORC1 is activated, make contact with three-way junctions where Lunapark resides. Inhibition of Lunapark ubiquitylation results in neurodevelopmental defects indicating that KLHL12-dependent ubiquitylation of Lunapark is required for normal growth and development.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Cullin/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Animales Modificados Genéticamente , Línea Celular Tumoral , Células HEK293 , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ubiquitinación , Pez CebraRESUMEN
Sleep is frequently altered in systemic infections as a component of sickness behavior in response to inflammation. Sleepiness in sickness behavior has been extensively investigated. Much less attention has instead been devoted to sleep and wake alterations in brain infections. Most of these, as other neuroinfections, are prevalent in sub-Saharan Africa. The present overview highlights the importance of this topic from both the clinical and pathogenetic points of view. Vigilance states and their regulation are first summarized, emphasizing that key nodes in this distributed brain system can be targeted by neuroinflammatory signaling. Sleep-wake changes in the parasitic disease human African trypanosomiasis (HAT) and its animal models are then reviewed and discussed. Experimental data have revealed that the suprachiasmatic nucleus, the master circadian pacemaker, and peptidergic cell populations of the lateral hypothalamus (the wake-promoting orexin neurons and the sleep-promoting melanin-concentrating hormone neurons) are targeted by African trypanosome infection. It is then discussed how prominent and disturbing are sleep changes in HIV/AIDS, also when the infection is cured with antiretroviral therapy. This recalls attention on the bidirectional interactions between sleep and immune system, including the specialized brain immune response of which microglial cells are protagonists. Sleep changes in an ancient viral disease, rabies, and in the emerging infection due to Zika virus which causes a congenital syndrome, are also dealt with. Altogether the findings indicate that sleep-wake regulation is targeted by brain infections caused by different pathogens and, although the relevant pathogenetic mechanisms largely remain to be clarified, these alterations differ from hypersomnia occurring in sickness behavior. Thus, brain infections point to the vulnerability of the neural network of sleep-wake regulation as a highly relevant clinical and basic science challenge.
Asunto(s)
Encéfalo/fisiología , Trastornos del Sueño-Vigilia/fisiopatología , Sueño/fisiología , Síndrome de Inmunodeficiencia Adquirida/fisiopatología , África/epidemiología , Humanos , Infecciones/patología , Microglía/fisiología , Neuronas/fisiología , Trastornos del Inicio y del Mantenimiento del Sueño/fisiopatología , Tripanosomiasis Africana/fisiopatología , Vigilia/fisiología , Infección por el Virus Zika/fisiopatologíaRESUMEN
The metallic impregnation invented by Camillo Golgi in 1873 has allowed the visualization of individual neurons in their entirety, leading to a breakthrough in the knowledge on the structure of the nervous system. Professor of Histology and of General Pathology, Golgi worked for decades at the University of Pavia, leading a very active laboratory. Unfortunately, most of Golgi's histological preparations are lost. The present contribution provides an account of the original slides on the nervous system from Golgi's laboratory available nowadays at the Golgi Museum and Historical Museum of the University of Pavia. Knowledge on the organization of the nervous tissue at the time of Golgi's observations is recalled. Notes on the equipment of Golgi's laboratory and the methodology Golgi used for his preparations are presented. Images of neurons from his slides (mostly from hippocampus, neocortex and cerebellum) are here shown for the first time together with some of Golgi's drawings. The sections are stained with the Golgi impregnation and Cajal stain. Golgi-impregnated sections are very thick (some more than 150 µm) and require continuous focusing during the microscopic observation. Heterogeneity of neuronal size and shape, free endings of distal dendritic arborizations, axonal branching stand out at the microscopic observation of Golgi-impregnated sections and in Golgi's drawings, and were novel findings at his time. Golgi also pointed out that the axon only originates from cell bodies, representing a constant and distinctive feature of nerve cells which distinguishes them from glia, and subserving transmission at a distance. Dendritic spines can be seen in some cortical neurons, although Golgi, possibly worried about artifacts, did not identify them. The puzzling intricacy of fully impregnated nervous tissue components offered to the first microscopic observations still elicit nowadays the emotion Golgi must have felt looking at his slides.
RESUMEN
Trypanosoma brucei (T. b.) gambiense is the parasite subspecies responsible for most reported cases of human African trypanosomiasis (HAT) or sleeping sickness. This severe infection leads to characteristic disruption of the sleep-wake cycle, recalling attention on the circadian timing system. Most animal models of the disease have been hitherto based on infection of laboratory rodents with the T. b. brucei subspecies, which is not infectious to humans. In these animal models, functional, rather than structural, alterations of the master circadian pacemaker, the hypothalamic suprachiasmatic nucleus (SCN), have been reported. Information on the SCN after infection with the human pathogenic T. b. gambiense is instead lacking. The present study was aimed at the examination of the SCN after T. b. gambiense infection of a susceptible rodent, the multimammate mouse, Mastomys natalensis, compared with T. b. brucei infection of the same host species. The animals were examined at 4 and 8 weeks post-infection, when parasites (T. b. gambiense or T. b. brucei) were detected in the brain parenchyma, indicating that the disease was in the encephalitic stage. Neuron and astrocyte changes were examined with Nissl staining, immunophenotyping and quantitative analyses. Interestingly, significant neuronal loss (about 30% reduction) was documented in the SCN during the progression of T. b. gambiense infection. No significant neuronal density changes were found in the SCN of T. b. brucei-infected animals. Neuronal cell counts in the hippocampal dentate gyrus of T. b. gambiense-infected M. natalensis did not point out significant changes, indicating that no widespread neuron loss had occurred in the brain. Marked activation of astrocytes was detected in the SCN after both T. b. gambiense and T. b. brucei infections. Altogether the findings reveal that neurons of the biological clock are highly susceptible to the infection caused by human pathogenic African trypanosomes, which have the capacity to cause permanent partial damage of this structure.
RESUMEN
BACKGROUND: Human African trypanosomiasis or sleeping sickness, caused by the parasite Trypanosoma brucei, leads to neuroinflammation and characteristic sleep/wake alterations. The relationship between the onset of these alterations and the development of neuroinflammation is of high translational relevance, but remains unclear. This study investigates the expression of interferon (IFN)-γ and IFN-inducible chemokine genes in the brain, and the levels of CXCL10 in the serum and cerebrospinal fluid prior to and during the encephalitic stage of trypanosome infection, and correlates these with sleep/wake changes in a rat model of the disease. METHODOLOGY/PRINCIPAL FINDINGS: The expression of genes encoding IFN-γ, CXCL9, CXCL10, and CXCL11 was assessed in the brain of rats infected with Trypanosoma brucei brucei and matched controls using semi-quantitative end-point RT-PCR. Levels of CXCL10 in the serum and cerebrospinal fluid were determined using ELISA. Sleep/wake states were monitored by telemetric recording. Using immunohistochemistry, parasites were found in the brain parenchyma at 14 days post-infection (dpi), but not at 6 dpi. Ifn-γ, Cxcl9, Cxcl10 and Cxcl11 mRNA levels showed moderate upregulation by 14 dpi followed by further increase between 14 and 21 dpi. CXCL10 concentration in the cerebrospinal fluid increased between 14 and 21 dpi, preceded by a rise in the serum CXCL10 level between 6 and 14 dpi. Sleep/wake pattern fragmentation was evident at 14 dpi, especially in the phase of wake predominance, with intrusion of sleep episodes into wakefulness. CONCLUSIONS/SIGNIFICANCE: The results show a modest increase in Cxcl9 and Cxcl11 transcripts in the brain and the emergence of sleep/wake cycle fragmentation in the initial encephalitic stage, followed by increases in Ifn-γ and IFN-dependent chemokine transcripts in the brain and of CXCL10 in the cerebrospinal fluid. The latter parameter and sleep/wake alterations could provide combined humoral and functional biomarkers of the early encephalitic stage in African trypanosomiasis.
Asunto(s)
Quimiocinas/sangre , Quimiocinas/líquido cefalorraquídeo , Encefalitis/parasitología , Sueño , Tripanosomiasis Africana/sangre , Tripanosomiasis Africana/líquido cefalorraquídeo , Animales , Biomarcadores , Encéfalo/parasitología , Encéfalo/patología , Interferón gamma/sangre , Interferón gamma/líquido cefalorraquídeo , Masculino , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Análisis de Regresión , Trypanosoma brucei bruceiRESUMEN
The paraventricular thalamic nucleus (PVT), the main component of the dorsal thalamic midline, receives multiple inputs from the brain stem and hypothalamus, and targets the medial prefrontal cortex, nucleus accumbens and amygdala. PVT has been implicated in several functions, especially adaptation to chronic stress, addiction behaviors and reward, mood, emotion. We here focus on the wiring and neuronal properties linking PVT with circadian timing and sleep/wake regulation, and their behavioral implications. PVT is interconnected with the master circadian pacemaker, the hypothalamic suprachiasmatic nucleus, receives direct and indirect photic input, is densely innervated by orexinergic neurons which play a key role in arousal and state transitions. Endowed with prominent wake-related Fos expression which is suppressed by sleep, and with intrinsic neuronal properties showing a diurnal oscillation unique in the thalamus, PVT could represent a station of interaction of thalamic and hypothalamic sleep/wake-regulatory mechanisms. PVT could thus play a strategic task by funneling into limbic and limbic-related targets circadian timing and state-dependent behavior information, tailoring it for cognitive performance and motivated behaviors.