Mutation in protein disulfide isomerase A3 causes neurodevelopmental defects by disturbing endoplasmic reticulum proteostasis.

Recessive gene mutations underlie many developmental disorders and often lead to disabling neurological problems. Here, we report identification of a homozygous c.170G>A (p.Cys57Tyr or C57Y) mutation in the gene coding for protein disulfide isomerase A3 (PDIA3, also known as ERp57), an enzyme that catalyzes formation of disulfide bonds in the endoplasmic reticulum, to be associated with syndromic intellectual disability. Experiments in zebrafish embryos show that PDIA3C57Y expression is pathogenic and causes developmental defects such as axonal disorganization as well as skeletal abnormalities. Expression of PDIA3C57Y in the mouse hippocampus results in impaired synaptic plasticity and memory consolidation. Proteomic and functional analyses reveal that PDIA3C57Y expression leads to dysregulation of cell adhesion and actin cytoskeleton dynamics, associated with altered integrin biogenesis and reduced neuritogenesis. Biochemical studies show that PDIA3C57Y has decreased catalytic activity and forms disulfide-crosslinked aggregates that abnormally interact with chaperones in the endoplasmic reticulum. Thus, rare disease gene variant can provide insight into how perturbations of neuronal proteostasis can affect the function of the nervous system.

Immunohistochemical characterisation of the adult Nothobranchius furzeri intestine.

Nothobranchius furzeri is emerging as an exciting vertebrate organism in the field of biomedicine, developmental biology and ecotoxicology research. Its short generation time, compressed lifespan and accelerated ageing make it a versatile model for longitudinal studies with high traceability. Although in recent years the use of this model has increased enormously, there is still little information on the anatomy, morphology and histology of its main organs. In this paper, we present a description of the digestive system of N. furzeri, with emphasis on the intestine. We note that the general architecture of the intestinal tissue is shared with other vertebrates, and includes a folding mucosa, an outer muscle layer and a myenteric plexus. By immunohistochemical analysis, we reveal that the mucosa harbours the same type of epithelial cells observed in mammals, including enterocytes, goblet cells and enteroendocrine cells, and that the myenteric neurons express neurotransmitters common to other species, such as serotonin, substance P and tyrosine hydroxylase. In addition, we detect the presence of a proliferative compartment at the base of the intestinal folds. The description of the normal intestinal morphology provided here constitutes a baseline information to contrast with tissue alterations in future lines of research assessing pathologies, ageing-related diseases or damage caused by toxic agents.

Organization of the Catecholaminergic System in the Short-Lived Fish Nothobranchius furzeri.

The catecholaminergic system has received much attention based on its regulatory role in a wide range of brain functions and its relevance in aging and neurodegenerative diseases. In the present study, we analyzed the neuroanatomical distribution of catecholaminergic neurons based on tyrosine hydroxylase (TH) immunoreactivity in the brain of adult Nothobranchius furzeri. In the telencephalon, numerous TH+ neurons were observed in the olfactory bulbs and the ventral telencephalic area, arranged as strips extending through the rostrocaudal axis. We found the largest TH+ groups in the diencephalon at the preoptic region level, the ventral thalamus, the pretectal region, the posterior tuberculum, and the caudal hypothalamus. In the dorsal mesencephalic tegmentum, we identified a particular catecholaminergic group. The rostral rhombencephalon housed TH+ cells in the locus coeruleus and the medulla oblongata, distributing in a region dorsal to the inferior reticular formation, the vagal lobe, and the area postrema. Finally, scattered TH+ neurons were present in the ventral spinal cord and the retina. From a comparative perspective, the overall organization of catecholaminergic neurons is consistent with the general pattern reported for other teleosts. However, N. furzeri shows some particular features, including the presence of catecholaminergic cells in the midbrain. This work provides a detailed neuroanatomical map of the catecholaminergic system of N. furzeri, a powerful aging model, also contributing to the phylogenetic understanding of one of the most ancient neurochemical systems.

Control of lysosomal-mediated cell death by the pH-dependent calcium channel RECS1.

Programmed cell death is regulated by the balance between activating and inhibitory signals. Here, we have identified RECS1 (responsive to centrifugal force and shear stress 1) [also known as TMBIM1 (transmembrane BAX inhibitor motif containing 1)] as a proapoptotic member of the TMBIM family. In contrast to other proteins of the TMBIM family, RECS1 expression induces cell death through the canonical mitochondrial apoptosis pathway. Unbiased screening indicated that RECS1 sensitizes cells to lysosomal perturbations. RECS1 localizes to lysosomes, where it regulates their acidification and calcium content, triggering lysosomal membrane permeabilization. Structural modeling and electrophysiological studies indicated that RECS1 is a pH-regulated calcium channel, an activity that is essential to trigger cell death. RECS1 also sensitizes whole animals to stress in vivo in Drosophila melanogaster and zebrafish models. Our results unveil an unanticipated function for RECS1 as a proapoptotic component of the TMBIM family that ignites cell death programs at lysosomes.

Acute intrastriatal administration of quinolinic acid affects the expression of the coat protein AP-2 and its interaction with membranes.

Clathrin-coated vesicle endocytosis is thought to be crucial for the maintenance of synaptic transmission and for the cell plasticity at the nervous system. In this study, we demonstrated that acute intrastriatal administration of quinolinic acid (QUIN), an agonist of the N-methyl-D: -aspartate receptor, induces a decrease of the coat protein AP-2 expression and affects their interaction with membranes. By western blot analysis we observed that at 24 h after QUIN intrastriatal injection, alpha1 subunit of AP-2 and alpha2, at lesser extent, were reduced in the striatal membranes. The decrease of both subunits expression was extended to 48 h after treatment, although the soluble proteins were mostly affected. Other areas of the brain were not affected by the treatment, except the cerebellum, where a significant increase of soluble AP-2 (both subunits) was observed at 48 h after injection. Another coat protein, as the phosphoprotein AP-180, was not affected by the injection of QUIN. We also confirmed that QUIN injection causes increasing loss of striatal neurons after the administration of the toxin. We concluded that QUIN may affect the endocytotic machinery of the striatum, by inducing changes in the AP-2 behaviour. Consequently, the internalization of NMDAR and/or AMPAR may be affected, by QUIN, contributing to the excitotoxic effect of the drug.

Changes in neural circuitry associated with depression at pre-clinical, pre-motor and early motor phases of Parkinson's disease.

Although Parkinson's Disease (PD) is mostly considered a motor disorder, it can present at early stages as a non-motor pathology. Among the non-motor clinical manifestations, depression shows a high prevalence and can be one of the first clinical signs to appear, even a decade before the onset of motor symptoms. Here, we review the evidence of early dysfunction in neural circuitry associated with depression in the context of PD, focusing on pre-clinical, pre-motor and early motor phases of the disease. In the pre-clinical phase, structural and functional changes in the substantia nigra, basal ganglia and limbic structures are already observed. Some of these changes are linked to motor compensation mechanisms while others correspond to pathological processes common to PD and depression and thus could underlie the appearance of depressive symptoms during the pre-motor phase. Studies of the early motor phase (less than five years post diagnosis) reveal an association between the extent of damage in different monoaminergic systems and the appearance of emotional disorders. We propose that the limbic loop of the basal ganglia and the lateral habenula play key roles in the early genesis of depression in PD. Alterations in the neural circuitry linked with emotional control might be sensitive markers of the ongoing neurodegenerative process and thus may serve to facilitate an early diagnosis of this disease. To take advantage of this, we need to improve the clinical criteria and develop biomarkers to identify depression, which could be used to determine individuals at risk to develop PD.

Immunization of rats against Fasciola hepatica using crude antigens conjugated with Freund's adjuvant or oligodeoxynucleotides.

Chronic Fasciola hepatica infection is correlated with the development of a T helper (Th2)-predominant immune response. To determine whether immunostimulatory CpG-containing oligodeoxynucleotides (CpG-ODN) or Freund's complete adjuvant (FCA), known to promote a Th1 (T helper 1) immune responses, could provide protection from F. hepatica infection, total homogenate (TH) of F. hepatica mixed with CpG-ODN or FCA were injected subcutaneously (s.c.) into Wistar rats. A F. hepatica-specific Th1-predominant immune response was induced with CpG-ODN or FCA in lymph nodes of immunized animals. Lymph node cells from TH-CpG-ODN or TH-FCA immunized rats showed increased antigen-specific proliferation with high levels of INFgamma, compared to lymphocytes from rats injected with TH alone. In contrast, these two groups of immunized animals did not modify IL-4 release by draining lymph node cells, when they were subsequently stimulated with TH in vitro. However, a significant reduction in the burden of flukes (76.7%) was only observed in rats immunized with TH-FCA. Conversely, immunization of rats with TH-CpG-ODN did not promote protection against the parasite. Therefore, even though CpG-ODNs and FCA induced Th1 type responses, only FCA provided a significant protection to rats infected with F. hepatica.

Mutant huntingtin affects endocytosis in striatal cells by altering the binding of AP-2 to membranes.

Clathrin-mediated endocytosis plays an important role in the maintenance of neuronal integrity in the synaptic terminals. Here we studied the effect of anomalous polyglutamine expansion in huntingtin on the interaction of coat proteins with membranes, in areas of mouse brain or in cultured striatal cells. We observed that this anomaly induces a redistribution of AP-2, but not other coat proteins, from the membrane to the cytosol in the striatum, and in the cultured striatal cells. It was also noted that huntingtin associates with AP-2, and that this association decreases due to the mutation in huntingtin. This decreased receptor-mediated endocytosis, measured by the internalization of transferrin in the mutated cells. It was also confirmed that huntingtin mutation made the cells more vulnerable to the action of quinolinic acid, with an increasing degradation of the AP-2 alpha subunits. On the basis of these results, we conclude that abnormal polyglutamine expansion in huntingtin affects clathrin-mediated endocytosis, and may be one of the pathogenic mechanisms of neurodegeneration.

Expression of coat proteins changes during postnatal development in selected areas of the rat brain.

It is well known that clathrin-mediated endocytosis is crucial for the normal functioning and integrity of neurons in the central nervous system. In this study we attempted to correlate the expression of coat proteins with development in different areas of rat brain. By Western blot, we studied the expression of AP-2, GGA1 and GGA2 in striatum, cerebellum, brain stem, cerebral cortex and hippocampus of newborn rats and during post-natal development; 5, 15, 30, 60, 90 or 150 days after birth. We observed that the expression of the α2 subunit of AP-2 increased substantially between the 15th and 30th day after birth in all areas studied, excepting the cerebellum and cortex. On the other hand, the expression of the α1 subunit does not change significantly during the development in any of the areas under study. We also noted that the expression of the µ2 subunit did not follow the pattern of α2 during development. In general terms, the expression of GGA1 and GGA2 followed a similar pattern to that of AP-2, although these proteins increased significantly in the cerebral cortex from the 15th day after birth. Moreover, presenilin-1, a protein associated with aging and neurodegeneration, shows an expression pattern similar to coat proteins in the striatum and cortex. These results suggest that proteins that conform the intracellular transport machinery in the brain cells seems to accompany development, according to the maturation of the different brain areas.