Typical bulbar ALS can be linked to GARS mutation.

Background: Amyotrophic lateral sclerosis is the most frequent motor neuron disorders (MND) in adults. The role of genetic factors is worldwide accepted, and currently, more than 30 genes have been linked to this disease. Genetics was also the matter of numerous studies in distal hereditary motor neuropathies (dHMN). GARS is classically linked to a predominant dHMN and, until now, no mutation has been described in GARS in other MND. Case Report: We report the case of a 70-year-old woman who developed a classical bulbar ALS phenotype. Owing to his familial history of ALS, a genetic screening was performed excluding the main genes linked to ALS and revealing a heterozygous missense mutation in GARS gene with a high probability of pathogenicity. Conclusion: This first description of mutation in GARS in ALS, extends once more the genetic overlap between ALS and other MND.

Causative Genes in Amyotrophic Lateral Sclerosis and Protein Degradation Pathways: a Link to Neurodegeneration.

Amyotrophic lateral sclerosis (ALS) is a disease caused by the degeneration of motor neurons (MNs) leading to progressive muscle weakness and atrophy. Several molecular pathways have been implicated, such as glutamate-mediated excitotoxicity, defects in cytoskeletal dynamics and axonal transport, disruption of RNA metabolism, and impairments in proteostasis. ALS is associated with protein accumulation in the cytoplasm of cells undergoing neurodegeneration, which is a hallmark of the disease. In this review, we focus on mechanisms of proteostasis, particularly protein degradation, and discuss how they are related to the genetics of ALS. Indeed, the genetic bases of the disease with the implication of more than 30 genes associated with familial ALS to date, together with the important increase in understanding of endoplasmic reticulum (ER) stress, proteasomal degradation, and autophagy, allow researchers to better understand the mechanisms underlying the selective death of motor neurons in ALS. It is clear that defects in proteostasis are involved in this type of cellular degeneration, but whether or not these mechanisms are primary causes or merely consequential remains to be clearly demonstrated. Novel cellular and animal models allowing chronic expression of mutant proteins, for example, are required. Further studies linking genetic discoveries in ALS to mechanisms of protein clearance will certainly be crucial in order to accelerate translational and clinical research towards new therapeutic targets and strategies.

Ptchd1 deficiency induces excitatory synaptic and cognitive dysfunctions in mouse.

Synapse development and neuronal activity represent fundamental processes for the establishment of cognitive function. Structural organization as well as signalling pathways from receptor stimulation to gene expression regulation are mediated by synaptic activity and misregulated in neurodevelopmental disorders such as autism spectrum disorder (ASD) and intellectual disability (ID). Deleterious mutations in the PTCHD1 (Patched domain containing 1) gene have been described in male patients with X-linked ID and/or ASD. The structure of PTCHD1 protein is similar to the Patched (PTCH1) receptor; however, the cellular mechanisms and pathways associated with PTCHD1 in the developing brain are poorly determined. Here we show that PTCHD1 displays a C-terminal PDZ-binding motif that binds to the postsynaptic proteins PSD95 and SAP102. We also report that PTCHD1 is unable to rescue the canonical sonic hedgehog (SHH) pathway in cells depleted of PTCH1, suggesting that both proteins are involved in distinct cellular signalling pathways. We find that Ptchd1 deficiency in male mice (Ptchd1-/y) induces global changes in synaptic gene expression, affects the expression of the immediate-early expression genes Egr1 and Npas4 and finally impairs excitatory synaptic structure and neuronal excitatory activity in the hippocampus, leading to cognitive dysfunction, motor disabilities and hyperactivity. Thus our results support that PTCHD1 deficiency induces a neurodevelopmental disorder causing excitatory synaptic dysfunction.

GABA/Glutamate synaptic pathways targeted by integrative genomic and electrophysiological explorations distinguish autism from intellectual disability.

Phenotypic and genetic heterogeneity is predominant in autism spectrum disorders (ASD), for which the molecular and pathophysiological bases are still unclear. Significant comorbidity and genetic overlap between ASD and other neurodevelopmental disorders are also well established. However, little is understood regarding the frequent observation of a wide phenotypic spectrum associated with deleterious mutations affecting a single gene even within multiplex families. We performed a clinical, neurophysiological (in vivo electroencephalography-auditory-evoked related potentials) and genetic (whole-exome sequencing) follow-up analysis of two families with known deleterious NLGN4X gene mutations (either truncating or overexpressing) present in individuals with ASD and/or with intellectual disability (ID). Complete phenotypic evaluation of the pedigrees in the ASD individuals showed common specific autistic behavioural features and neurophysiological patterns (abnormal MisMatch Negativity in response to auditory change) that were absent in healthy parents as well as in family members with isolated ID. Whole-exome sequencing in ASD patients from each family identified a second rare inherited genetic variant, affecting either the GLRB or the ANK3 genes encoding NLGN4X interacting proteins expressed in inhibitory or in excitatory synapses, respectively. The GRLB and ANK3 mutations were absent in relatives with ID as well as in control databases. In summary, our findings provide evidence of a double-hit genetic model focused on excitatory/inhibitory synapses in ASD, that is not found in isolated ID, associated with an atypical in vivo neurophysiological pattern linked to predictive coding.

Association study of the NF1 gene and autistic disorder.

Neurofibromatosis type 1 (NF1) is increased about 150-fold in autistic patients. The aim of this study was to test for an association between the NF1 locus and autistic disorder. The allele distributions of three markers of the NF1 gene were studied in 85 autistic patients and 90 controls. No differences in allele distributions were observed. However, we found a new allele (allele 5) of the GXAlu marker in four autistic patients. Allele 5 was absent in a larger control population (213 individuals). The patients with allele 5 had a more severe clinical picture, mainly in the fields of motility and tonus. Our preliminary results suggest that the NF1 region is not a major susceptibility locus for autism. However, the GXAlu marker of the NF1 gene appears as a possible candidate for a susceptibility locus in a small subgroup of severely affected autistic patients. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 88:729-732, 1999.

Cell lines with extended in vitro growth potential from human renal proximal tubule: characterization, response to inducers, and comparison with established cell lines.

Few model systems exist for the study of injury to human renal proximal tubule epithelium. Optimized differentiated human renal epithelial cell lines with extended in vitro growth potential would provide an alternative model system to primary culture or other available non-human mammalian kidney cell lines. For this purpose, human renal tubule epithelial cells were isolated from normal kidney cortex and exposed in culture to a hybrid immortalizing virus, adenovirus 12-SV40. Cell lines were developed by limiting dilution, and three selected cell lines were screened for growth pattern, production of immortalizing virus, tumorigenicity, and ploidy. Cell lines were also monitored for response to inducer agents and matrix factors and were screened for expression of biochemical properties and differentiation markers of renal epithelium. All three are nonproducers of the immortalizing virus and are nontumorigenic. They grow in monolayer, have intermediate growth kinetics, and express markers of renal proximal tubular epithelium by immunohistochemistry. They also express biochemical properties comparable to other widely used proximal tubular cell lines including LLC-RK1, OK, and HK-2 and comparable to human tubular cells in stable culture. Growth medium containing low levels of fetal calf serum, or epidermal growth factor combined with parathyroid hormone, produced optimal growth characteristics, brush border enzyme expression, biochemical properties, and glucose transport in a selected cell line. The addition of dimethyl sulfoxide allows maintenance in morphologically intact monolayers for prolonged periods. These cell lines should be useful model systems for the study of human renal proximal tubular injury or disease.

Comparative impact of cephaloridine on glutathione and related enzymes in LLC-PK1, LLC-RK1, and primary cultures of rat and rabbit proximal tubule cells.

Among kidney tubular epithelial cell types, proximal tubule cells are one of the major renal targets for xenobiotics. Several in vitro culture models have been proposed for use of proximal tubule cells for in vitro pharmacotoxicology studies. This paper reports a comparative study of the response to cephaloridine exposure of two established cell lines from pig (LLC-PK1) and rabbit (LLC-RK1) kidneys and primary cultures of rat and rabbit proximal tubule cells. These cultured cells were first compared for their levels of activity of alpha-methylglucopyranoside transport, alkaline phosphatase, succinate dehydrogenase, and NADPH cytochrome c reductase, their glutathione-dependent activity levels, and their adenylate cyclase response pattern to stimulation by PTH and AVP. The results presented show major phenotypic differences between these four cellular models. The differences observed in glutathione-dependent mechanism activities and regulation may in part be responsible for the variability of the responses of these four cellular models when exposed to cephaloridine.

Impact of cephaloridine on glutathione and related enzymes: comparison of in vivo and in vitro rat models.

The aim of this study was to investigate the early effects of cephaloridine (CPH) on glutathione-dependent phase II detoxification in the rat proximal tubular cell and to find an in vitro alternative to the in vivo model. The in vivo study was conducted in three groups of rats which received CPH at doses of 250, 500 or 750 mg/kg per day for 3 days, while another group received 500 mg/kg as a single dose. For the in vitro study, rat renal proximal tubular cultured cells were exposed to CPH at concentrations of 0.3, 0.6, 1, 1.7 mM for 24, 48 and 72 h. Glutathione-dependent detoxification was evaluated in vivo and in vitro on the basis of total intracellular glutathione (GSH), glutathione S-transferase (GST) and glutathione peroxidase (GPX). Glutathione reductase (GRED) and GST mRNA levels were also determined. Results of in vivo and in vitro models were comparable in terms of the early increase of GSH, GST and GRED. This increase had a bell-shaped dose-response with a maximum at 500 mg/kg in vivo and 1 mM in vitro. Beyond these doses, GSH and its dependent enzyme levels decreased, associated with cytotoxicity in vitro and renal insufficiency in vivo. The increased GST activity was associated with an increased level of GST7 in vivo and a markedly increased level of GST1-2 in vitro. We concluded that the in vitro model can be used as an alternative to animal experimentation to study glutathione-dependent detoxication. Low cytotoxic doses of CPH induced an early increase of glutathione phase II-dependent detoxification enzymes.

Effects of the medium HCO3-/CO2 buffer system on differentiation and intermediary metabolism properties of rabbit proximal tubule cells in primary culture.

In vivo, bicarbonate can affect proximal tubule intermediary metabolism, including gluconeogenesis, ammoniagenesis and maintenance of the mitochondrial substrate supply. In vitro, rabbit proximal tubule cells (RPTC) in primary culture revert from gluconeogenesis to glycolysis and their mitochondrial metabolism remains lower than in vivo. To determine whether the bicarbonate buffer system could have an effect on these deregulations, RPTC in primary culture grown in the absence of insulin and glucose in the culture medium were developed either with the standard sodium bicarbonate buffer with 5% CO2 or with a Hepes hydrogen ion buffer in the presence of 0.5% CO2. Duration of the bicarbonate-free cultures was increased until at least day 17 after seeding, compared with day 11 in bicarbonate-buffered cultures. As could be expected, succinate dehydrogenase activity remained stable as a function of time in bicarbonate-free cultures while an early marked decrease of this activity occurred from seeding in cultures developed in the presence of bicarbonate buffer. Compared to bicarbonate-buffered cells, higher phosphoenolpyruvate carboxykinase activity concomitant with lower intracellular lactate dehydrogenase activity was observed in cultures developed in the absence of bicarbonate, which is indicative of closer carbohydrate metabolism orientation to the in vivo situation for RPTC. Immunofluorescence staining of RPTC with monoclonal antibodies directed to neutral endopeptidase (NEP), and dipeptidyl-peptidase IV (DPP II) showed similar extensive labelling with DPP and NEP in both culture conditions. Confocal microscopy analysis of NEP subcellular distribution, showed exclusive targetting of NEP to the apical plasma membranes. In both models, cAMP production was stimulated by parathyroid hormone and unaffected by arginine vasopressin. In conclusion, bicarbonate withdrawal from the culture medium (without changing the pH of the medium) allows a marked improvement of mitochondrial capacity and carbohydrate metabolism pattern without any loss of differentiated properties.

Some milestones in in vitro organ Toxicity Assessment. The Kidney as a Case Study.

The expression of target organ toxicity ranges from subtle abnormalities of cellular organelles to permanent loss of organ function. The selective targeting of chemicals for discrete regions and cell types of a given organ is frequently due (besides some pharmacodynamic mechanisms) to the fact that target cells may express unique biochemical or functional characteristics predisposing them to chemically induced injury. In vitro models commonly used in target organ toxicity tests include perfused organ preparations, isolated tissue preparations, single-cell suspensions and tissue culture systems. Although these systems have proved their usefulness for acute toxicity tests, there is still a great need for in vitro models to be used for chronic toxicity tests. Among the systems listed above, the single-cell culture technique may be adapted to long-term study requirements. The example of kidney proximal tubules is taken to illustrate the necessity for extensive characterization of the actual capacities of the models in term of phenotypic profiling, energy status, drug detoxication activities, specific transport systems and organ-specific differentiated functions. LLC-PK1, LLC-RK1, NRK and OK cell lines are compared with primary cultures of rat, rabbit and human proximal tubule cells. The importance of the cell culture environment on the cell phenotypic profile, and its subsequent response pattern to toxicant exposure, are described using gentamicin and platinum derivatives as examples. In terms of experimental strategy, choice of cell type, choice of species of origin, choice of doses, choice of duration, continuous or discontinuous exposure, and whether to study the recovery phase, are crucial issues for designing models mimicking more closely the in vivo situation. The identification of relevant endpoints, allowing discrimination between general cell toxicity and specific organ toxicity, has not been sufficiently explored in vitro. Scientifically based endpoints referring to the background studies conducted by biochemists or physiologists should be selected and included in experimental designs dealing with organ toxicology in vitro. Conceptually, relevant specific target-organ toxicity could be investigated by the use of multiple cell types and by analysis of the difference in concentration between the cytotoxic concentration threshold and the specific endpoint alteration threshold.