The genomic landscape across 474 surgically accessible epileptogenic human brain lesions.

Understanding the exact molecular mechanisms involved in the aetiology of epileptogenic pathologies with or without tumour activity is essential for improving treatment of drug-resistant focal epilepsy. Here, we characterize the landscape of somatic genetic variants in resected brain specimens from 474 individuals with drug-resistant focal epilepsy using deep whole-exome sequencing (>350×) and whole-genome genotyping. Across the exome, we observe a greater number of somatic single-nucleotide variants in low-grade epilepsy-associated tumours (7.92 ± 5.65 single-nucleotide variants) than in brain tissue from malformations of cortical development (6.11 ± 4 single-nucleotide variants) or hippocampal sclerosis (5.1 ± 3.04 single-nucleotide variants). Tumour tissues also had the largest number of likely pathogenic variant carrying cells. low-grade epilepsy-associated tumours had the highest proportion of samples with one or more somatic copy-number variants (24.7%), followed by malformations of cortical development (5.4%) and hippocampal sclerosis (4.1%). Recurring somatic whole chromosome duplications affecting Chromosome 7 (16.8%), chromosome 5 (10.9%), and chromosome 20 (9.9%) were observed among low-grade epilepsy-associated tumours. For germline variant-associated malformations of cortical development genes such as TSC2, DEPDC5 and PTEN, germline single-nucleotide variants were frequently identified within large loss of heterozygosity regions, supporting the recently proposed 'second hit' disease mechanism in these genes. We detect somatic variants in 12 established lesional epilepsy genes and demonstrate exome-wide statistical support for three of these in the aetiology of low-grade epilepsy-associated tumours (e.g. BRAF) and malformations of cortical development (e.g. SLC35A2 and MTOR). We also identify novel significant associations for PTPN11 with low-grade epilepsy-associated tumours and NRAS Q61 mutated protein with a complex malformation of cortical development characterized by polymicrogyria and nodular heterotopia. The variants identified in NRAS are known from cancer studies to lead to hyperactivation of NRAS, which can be targeted pharmacologically. We identify large recurrent 1q21-q44 duplication including AKT3 in association with focal cortical dysplasia type 2a with hyaline astrocytic inclusions, another rare and possibly under-recognized brain lesion. The clinical-genetic analyses showed that the numbers of somatic single-nucleotide variant across the exome and the fraction of affected cells were positively correlated with the age at seizure onset and surgery in individuals with low-grade epilepsy-associated tumours. In summary, our comprehensive genetic screen sheds light on the genome-scale landscape of genetic variants in epileptic brain lesions, informs the design of gene panels for clinical diagnostic screening and guides future directions for clinical implementation of epilepsy surgery genetics.

Increased paternal corticosterone exposure influences offspring behaviour and expression of urinary pheromones.

BACKGROUND: Studies have shown that paternal stress prior to conception can influence the innate behaviours of their offspring. The evolutionary impacts of such intergenerational effects are therefore of considerable interest. Our group previously showed in a model of daily stress that glucocorticoid treatment of adult male mouse breeders prior to conception leads to increased anxiety-related behaviours in male offspring. Here, we aimed to understand the transgenerational effects of paternal stress exposure on the social behaviour of progeny and its potential influence on reproductive success. RESULTS: We assessed social parameters including social reward, male attractiveness and social dominance, in the offspring (F1) and grand-offspring (F2). We report that paternal corticosterone treatment was associated with increased display of subordination towards other male mice. Those mice were unexpectedly more attractive to female mice while expressing reduced levels of the key rodent pheromone Darcin, contrary to its conventional role in driving female attraction. We investigated the epigenetic regulation of major urinary protein (Mup) expression by performing the first Oxford Nanopore direct methylation of sperm DNA in a mouse model of stress, but found no differences in Mup genes that could be attributed to corticosterone-treatment. Furthermore, no overt differences of the prefrontal cortex transcriptome were found in F1 offspring, implying that peripheral mechanisms are likely contributing to the phenotypic differences. Interestingly, no phenotypic differences were observed in the F2 grand-offspring. CONCLUSIONS: Overall, our findings highlight the potential of moderate paternal stress to affect intergenerational (mal)adaptive responses, informing future studies of adaptiveness in rodents, humans and other species.

Ganglioglioma with adverse clinical outcome and atypical histopathological features were defined by alterations in PTPN11/KRAS/NF1 and other RAS-/MAP-Kinase pathway genes.

Exome-wide sequencing studies recently described PTPN11 as a novel brain somatic epilepsy gene. In contrast, germline mutations of PTPN11 are known to cause Noonan syndrome, a multisystem disorder characterized by abnormal facial features, developmental delay, and sporadically, also brain tumors. Herein, we performed a deep phenotype-genotype analysis of a comprehensive series of ganglioglioma (GG) with brain somatic alterations of the PTPN11/KRAS/NF1 genes compared to GG with common MAP-Kinase signaling pathway alterations, i.e., BRAFV600E. Seventy-two GG were submitted to whole exome sequencing and genotyping and 84 low grade epilepsy associated tumors (LEAT) to DNA-methylation analysis. In 28 tumours, both analyses were available from the same sample. Clinical data were retrieved from hospital files including disease onset, age at surgery, brain localization, and seizure outcome. A comprehensive histopathology staining panel was available in all cases. We identified eight GG with PTPN11 alterations, copy number variant (CNV) gains of chromosome 12, and the commonality of additional CNV gains in NF1, KRAS, FGFR4 and RHEB, as well as BRAFV600E alterations. Histopathology revealed an atypical glio-neuronal phenotype with subarachnoidal tumor spread and large, pleomorphic, and multinuclear cellular features. Only three out of eight patients with GG and PTPN11/KRAS/NF1 alterations were free of disabling-seizures 2 years after surgery (38% had Engel I). This was remarkably different from our series of GG with only BRAFV600E mutations (85% had Engel I). Unsupervised cluster analysis of DNA methylation arrays separated these tumours from well-established LEAT categories. Our data point to a subgroup of GG with cellular atypia in glial and neuronal cell components, adverse postsurgical outcome, and genetically characterized by complex alterations in PTPN11 and other RAS-/MAP-Kinase and/or mTOR signaling pathways. These findings need prospective validation in clinical practice as they argue for an adaptation of the WHO grading system in developmental, glio-neuronal tumors associated with early onset focal epilepsy.

Neuropathology and epilepsy surgery.

PURPOSE OF REVIEW: Neurosurgical treatment of patients suffering from drug-resistant focal epilepsy is recognized as a successful, yet underutilized medical treatment option. By searching PubMed for articles published between January 2020 and September 2021 with the broad search terms 'neuropathology' AND 'epilepsy surgery', this review highlights the active field of etiology-based epilepsy research in human tissue. RECENT FINDINGS: All papers addressing the most common epileptogenic human brain disease entities, i.e. focal cortical dysplasia (FCD), brain tumors or hippocampal sclerosis, and written in English language were eligible for our review. We can conclude from this review that etiology-based studies are of foremost interest for (1) the development of prediction models for postsurgical seizure outcome; (2) decipher genetic and molecular alterations to better define disease entities and underlying molecular pathomechanisms, and (3) the translation of human tissue-derived biomarker into clinically useful diagnostics or novel therapeutic targets in the near future. SUMMARY: Highlighting FCD brain somatic gain-of-function variants in mammalian target of Rapamycin are a leading pathway to better classify FCD. An integrated genotype-phenotype analysis enables to classify the broad spectrum of low-grade and epilepsy-associated brain tumors. Further DNA-methylation-based disease classification will increase the mechanistic understanding and diagnostic precision of difficult to classify pathologies in the future.

Age-dependent increase of perineuronal nets in the human hippocampus and precocious aging in epilepsy.

OBJECTIVE: Perineuronal nets (PNN) are specialized extracellular matrix (ECM) components of the central nervous system, frequently accumulating at the surface of inhibitory GABAergic interneurons. While an altered distribution of PNN has been observed in neurological disorders including Alzheimer's disease, schizophrenia and epilepsy, their anatomical distribution also changes during physiological brain maturation and aging. Such an age-dependent shift was experimentally associated also with hippocampal engram formation during brain maturation. Our aim was to histopathologically assess PNN in the hippocampus of adult and pediatric patients with temporal lobe epilepsy (TLE) compared to age-matched post-mortem control subjects and to compare PNN-related changes with memory impairment observed in our patient cohort. METHODS: Sixty-six formalin-fixed and paraffin-embedded tissue specimens of the human hippocampus were retrieved from the European Epilepsy Brain Bank. Twenty-nine patients had histopathologically confirmed hippocampal sclerosis (HS), and eleven patients suffered from TLE without HS. PNN were immunohistochemically visualized using an antibody directed against aggrecan and manually counted from hippocampus subfields and the subiculum. RESULTS: PNN density increased with age in both human controls and TLE patients. However, their density was significantly higher in all HS patients compared to age-matched controls. Intriguingly, TLE patients presented presurgically with better memory when their hippocampal PNN density was higher (p < 0.05). SIGNIFICANCE: Our results were compatible with age-dependent ECM specialization in the human hippocampus and its precocious aging in the epileptic condition. These observations confirm recent experimental animal models and also support the notion that PNN play a role in memory formation in the human brain. PLAIN LANGUAGE SUMMARY: "Perineuronal nets" (PNN) are a specialized compartment of the extracellular matrix (ECM), especially surrounding highly active neurons of the mammalian brain. There is evidence that PNN play a role in memory formation, brain maturation, and in some pathologies like Alzheimer's disease, schizophrenia or epilepsy. In this study, we investigated the role of PNN in patients suffering from drug-resistant focal epilepsy compared to controls. We found that with increasing age, more neurons are surrounded by PNN. Similarly, all epilepsy patients but especially patients with better memory performance also had more PNN. This study raises further interest in studying ECM molecules in the human brain under physiological and pathophysiological conditions.

Paternal early life stress exerts intergenerational effects on male C57Bl/6J offspring risk-taking behaviors and predator scent-induced c-Fos expression.

Paternal preconceptional health factors, such as exposures to stress, diet and exercise, have been found to significantly influence offspring phenotypes in a range of animal models. Preclinical studies have provided evidence that paternal stress is associated with increased stress responsivity and anxiety-related traits, particularly in male offspring. It was previously reported that a paternal history of maternal separation (MS) led to male offspring (PatMS) displaying reduced cautious behavior during exploration of a novel environment. The neural basis for that absence of behavioral moderation is unclear. Here, we investigated the adaptive behavioral responses of control and PatMS male offspring in the predator odor risk-assessment task (PORT). PatMS mice failed to moderate their behaviors in the presence of a predator odor 2,4,5-trimethylthiazoline (TMT). c-Fos mapping revealed reduced cellular activation in fear-regulating brain regions of PatMS mice, such as in the cingulate cortex, dentate gyrus of the hippocampus and the basolateral amygdala. Expression of the paternally imprinted gene Grb10 (previously identified as a key molecular regulator of risk-taking behavior) was unaltered in PatMS mice. However, other paternal imprinted genes such as Igf2 and PEG3 were differentially expressed in PatMS mice. Overall, our study provides the first evidence of an intergenerational influence of preconceptional paternal stress exposure on offspring brain zunction relevant to risk-taking behavior, which is also independent of Grb10 gene expression.

Deep histopathology genotype-phenotype analysis of focal cortical dysplasia type II differentiates between the GATOR1-altered autophagocytic subtype IIa and MTOR-altered migration deficient subtype IIb.

Focal cortical dysplasia type II (FCDII) is the most common cause of drug-resistant focal epilepsy in children. Herein, we performed a deep histopathology-based genotype-phenotype analysis to further elucidate the clinico-pathological and genetic presentation of FCDIIa compared to FCDIIb. Seventeen individuals with histopathologically confirmed diagnosis of FCD ILAE Type II and a pathogenic variant detected in brain derived DNA whole-exome sequencing or mTOR gene panel sequencing were included in this study. Clinical data were directly available from each contributing centre. Histopathological analyses were performed from formalin-fixed, paraffin-embedded tissue samples using haematoxylin-eosin and immunohistochemistry for NF-SMI32, NeuN, pS6, p62, and vimentin. Ten individuals carried loss-of-function variants in the GATOR1 complex encoding genes DEPDC5 (n = 7) and NPRL3 (n = 3), or gain-of-function variants in MTOR (n = 7). Whereas individuals with GATOR1 variants only presented with FCDIIa, i.e., lack of balloon cells, individuals with MTOR variants presented with both histopathology subtypes, FCDIIa and FCDIIb. Interestingly, 50% of GATOR1-positive cases showed a unique and predominantly vacuolizing phenotype with p62 immunofluorescent aggregates in autophagosomes. All cases with GATOR1 alterations had neurosurgery in the frontal lobe and the majority was confined to the cortical ribbon not affecting the white matter. This pattern was reflected by subtle or negative MRI findings in seven individuals with GATOR1 variants. Nonetheless, all individuals were seizure-free after surgery except four individuals carrying a DEPDC5 variant. We describe a yet underrecognized genotype-phenotype correlation of GATOR1 variants with FCDIIa in the frontal lobe. These lesions were histopathologically characterized by abnormally vacuolizing cells suggestive of an autophagy-altered phenotype. In contrast, individuals with FCDIIb and brain somatic MTOR variants showed larger lesions on MRI including the white matter, suggesting compromised neural cell migration.

Experimental Epileptogenesis in a Cell Culture Model of Primary Neurons from Rat Brain: A Temporal Multi-Scale Study.

Understanding seizure development requires an integrated knowledge of different scales of organization of epileptic networks. We developed a model of "epilepsy-in-a-dish" based on dissociated primary neuronal cells from neonatal rat hippocampus. We demonstrate how a single application of glutamate stimulated neurons to generate spontaneous synchronous spiking activity with further progression into spontaneous seizure-like events after a distinct latency period. By computational analysis, we compared the observed neuronal activity in vitro with intracranial electroencephalography (EEG) data recorded from epilepsy patients and identified strong similarities, including a related sequence of events with defined onset, progression, and termination. Next, a link between the neurophysiological changes with network composition and cellular structure down to molecular changes was established. Temporal development of epileptiform network activity correlated with increased neurite outgrowth and altered branching, increased ratio of glutamatergic over GABAergic synapses, and loss of calbindin-positive interneurons, as well as genome-wide alterations in DNA methylation. Differentially methylated genes were engaged in various cellular activities related to cellular structure, intracellular signaling, and regulation of gene expression. Our data provide evidence that a single short-term excess of glutamate is sufficient to induce a cascade of events covering different scales from molecule- to network-level, all of which jointly contribute to seizure development.

A Preclinical Model of Computerized Cognitive Training: Touchscreen Cognitive Testing Enhances Cognition and Hippocampal Cellular Plasticity in Wildtype and Alzheimer's Disease Mice.

With the growing popularity of touchscreen cognitive testing in rodents, it is imperative to understand the fundamental effects exposure to this paradigm can have on the animals involved. In this study, we set out to assess hippocampal-dependant learning in the APP/PS1 mouse model of Alzheimer's disease (AD) on two highly translatable touchscreen tasks - the Paired Associate Learning (PAL) task and the Trial Unique Non-Matching to Location (TUNL) task. Both of these tests are based on human tasks from the Cambridge Neuropsychological Test Automated Battery (CANTAB) and are sensitive to deficits in both mild cognitive impairment (MCI) and AD. Mice were assessed for deficits in PAL at 9-12 months of age, then on TUNL at 8-11 and 13-16 months. No cognitive deficits were evident in APP/PS1 mice at any age, contrary to previous reports using maze-based learning and memory tasks. We hypothesized that daily and long-term touchscreen training may have inadvertently acted as a cognitive enhancer. When touchscreen-tested mice were assessed on the Morris water maze, they showed improved task acquisition compared to naïve APP/PS1 mice and wild-type (WT) littermate controls. In addition, we show that touchscreen-trained WT and APP/PS1 mice show increased cell proliferation and immature neuron numbers in the dentate gyrus compared to behaviorally naïve WT and APP/PS1 mice. This result indicates that the touchscreen testing paradigm could improve cognitive performance, and/or mask an impairment, in experimental mouse models. This touchscreen-induced cognitive enhancement may involve increased neurogenesis, and possibly other forms of cellular plasticity. This is the first study to show increased numbers of proliferating cells and immature neurons in the hippocampus following touchscreen testing, and that touchscreen training can improve cognitive performance in maze-based spatial navigation tasks. This potential for touchscreen testing to induce cognitive enhancement, or other phenotypic shifts, in preclinical models should be considered in study design. Furthermore, touchscreen-mediated cognitive enhancement could have therapeutic implications for cognitive disorders.

Fungal beta-glucans as adjuvants for treating cancer patients - A systematic review of clinical trials.

BACKGROUND & AIMS: Fungal ß-glucans have been considered as biological response modifiers (BRMs) promoting stimulation of immune system according to numerous scientific publications performed in vitro and in vivo. Some clinical trials involving such compounds started to be published since 1980's. This systematic review aimed to compile and compare clinical studies using these ß-glucans as adjuvants on patients undergoing cancer treatment. Healthy subjects and ß-glucans from other sources were excluded. METHODS: It was developed according to PRISMA-P guidelines (PROSPERO registered n. CRD42020151539), using PICO criteria and the following databases: PubMed, Scielo and LILACS. RESULTS: We found 1018 articles and after removing duplicated records, select by title/abstract and full-text, only 9 studies remained and 7 more were manually added, totalizing 16 trials involving 1650 patients, with arm sizes varying from 9 until 200 patients. The selected studies (published since 1992-2018) included subjects with diagnosis of 9 types of cancer. The studies used different sources of ß-glucans, such as yeast (Saccharomyces cerevisiae), mushrooms (Lentinula edodes and Schizophyllum commune) and non-described fungal sources. CONCLUSIONS: It was observed that the administration of ß-glucan is safe and well-tolerated. Most of the trials pointed that concomitant administration of ß-glucan with chemo or radiotherapy reduced the immune depression caused by such treatments and/or accelerated the recovery of white blood cells counts. However, some articles also commented that no statistical difference was encountered between ß-glucan treated vs. control groups, which gives a controversial conclusion about the ß-glucan effects. The great diversity among the methodology studies and insufficient information was an impeditive for achieving profound statistical analysis, therefore a narrative report of the included studies was performed indicating that further evidences are required to determine the efficacy of this adjuvant in the cancer treatment.

Evaluation of PD-L1 and tumor infiltrating lymphocytes in paired pretreatment biopsies and post neoadjuvant chemotherapy surgical specimens of breast carcinoma.

Herein it was evaluated the impact of PD-L1 immunohistochemical expression and stromal tumor-infiltrating lymphocyte (sTIL) counts in pretreatment needle core biopsy on response to neoadjuvant chemotherapy (NACT) for patients with breast carcinomas (BC). In 127 paired pre- and post-NACT BC specimens, immunohistochemical expression of PD-L1 was evaluated in stroma and in neoplastic cells. In the same samples sTILs were semi-quantified in tumor stroma. Post-NACT specimens were histologically rated as having residual cancer burden (RCB of any degree), or with complete pathological response (pCR). PD-L1 expression and higher sTIL counts were associated with histological grade 3 BC. PD-L1 expression was also associated with the non-luminal-HER2+ and triple negative immunohistochemical profiles of BC. Pathological complete response was associated with histological grade 3 tumors, and with the non-luminal-HER2+ and triple negative profiles. Additionally, our results support an association between PD-L1 expression and pCR to NACT. It was also observed that there is a trend to reduction of sTIL counts in the post-NACT specimens of patients with pCR. Of note, PD-L1 was expressed in half of the hormone receptor positive cases, a finding that might expand the potential use of immune checkpoint inhibitors for BC patients.

Effects of Ethanol Exposure During Adolescence or Adulthood on Locomotor Sensitization and Dopamine Levels in the Reward System.

Behavioral sensitization is a process of neuroadaptation characterized by a gradual increase in motor behaviors. The major neural substrates involved in the behavioral sensitization lie on the dopaminergic mesocorticolimbic pathway, which is still under development during adolescence. To investigate age-differences in ethanol behavioral sensitization and dopamine levels in distinct brain regions of the reward system, adolescent and adult mice were repeatedly pretreated with saline or ethanol (2.0 g/kg i.p.) during 15 consecutive days and challenged with saline or ethanol 5 days after pretreatment. Dopamine and its metabolites were measured in tissue samples of the prefrontal cortex (PFC), nucleus accumbens (NAc) and striatum by HPLC analysis. While repeated ethanol administration resulted in the development of locomotor sensitization in both adult and adolescent mice, only the adults expressed sensitization to a subsequent ethanol challenge injection. Neurochemical results showed reduced dopamine levels in adolescents compared to adults. Specifically, mice pretreated with ethanol during adolescence displayed lower dopamine levels in the PFC compared to the respective adult group in response to an ethanol challenge injection, and preadolescent mice exhibited lower dopamine levels in the NAc following an acute ethanol treatment compared to adults. These findings suggest that adolescent mice are not only less sensitive to the expression of ethanol-induced sensitization than adults, but also show lower dopamine content after ethanol exposition in the PFC and NAc.

Preconceptual paternal environmental stimulation alters behavioural phenotypes and adaptive responses intergenerationally in Swiss mice.

Experimental research has recently revealed that paternal environmental conditions can influence the offspring phenotype through epigenetic mechanisms. However, it is unclear whether these effects impact adaptive responses in the offspring. Environmental enrichment (EE) is a well-established paradigm that promotes neural plasticity. We investigated whether EE in male mice could modify behaviours that are highly relevant for determining adaptive fitness, i.e. spatial memory, attractiveness and social dominance, in the offspring of outbred mice. Male Swiss mice were housed in either EE or standard housing from post-weaning to adulthood before breeding for offspring. Their offspring were raised in standard housing until adulthood then assessed for behavioural, physiological and molecular parameters. F0 male mice exposed to EE had lower body weight, higher adrenal, spleen and hippocampal weights, better novelty processing and spatial learning, greater hippocampal BDNF levels, and higher social dominance. Unexpectedly, their male offspring (F1) showed spatial memory impairment, lowered social dominance and were less attractive to receptive females, compared to controls. These ethologically relevant measures suggest a maladaptive response in the male F1 offspring. Interestingly, when separate cohorts of male F1 offspring of standard housing or EE fathers were exposed to 8-day EE protocol during adulthood, differences in spatial memory and attractiveness to receptive females were no longer observed between them. These results provide evidence that the paternal environment can influence the offspring's adaptiveness.

Cocaine-induced behavioral sensitization is greater in adolescent than in adult mice and heightens cocaine-induced conditioned place preference in adolescents.

Adolescents are more sensitive than adults to the neural and behavioral effects of psychostimulants, and exhibit greater vulnerability to drug abuse, dependence or relapse into these conditions. We have reported that cocaine pretreatment during adolescence promotes the expression of behavioral sensitization to a greater extent than when the pretreatment occurs at adulthood. Behavioral sensitization has been associated to the transition from drug use to addiction and is postulated to indicate heightened sensitivity to the appetitive motivational effects of drugs. The relationship between behavioral sensitization and conventional measures of drug reward, such as conditioned place preference (CPP), has yet to be thoroughly investigated, and little is known about age-related differences in this phenomenon. The present study tested cocaine-induced CPP in adolescent and adult mice exposed to cocaine (or vehicle) pretreatment, either in an intermittent or "binge" (i.e., heavy cocaine use on a single occasion, which increases the likelihood of experiencing cocaine-related problems) fashion. Cocaine administration induced behavioral sensitization to a greater extent in adolescent than in adult mice. Cocaine-induced CPP was fairly similar in vehicle pretreated adolescent and adult mice, yet greater in adolescent vs. adults after cocaine-induced sensitization. The results confirmed the higher sensitivity of adolescent mice to cocaine-induced behavioral sensitization and suggest its association with greater sensitivity to cocaine's rewarding effects.

Effects of environmental enrichment upon ethanol-induced conditioned place preference and pre-frontal BDNF levels in adolescent and adult mice.

Environmental enrichment (EE) provides a non-pharmacological tool to alter drug-induced reward, yet its effects on ethanol-induced reward remain controversial. We analyzed adolescent vs. adult (mice) differences in the influence of EE on ethanol-induced conditioned place preference (CPP). The effects of these treatments on brain-derived neurotrophic factor (BDNF) levels in the prefrontal cortex were examined in a separate group of animals. Ethanol-induced CPP was found in adults, and it was similar in EE and in animals reared under standard housing conditions (SC). Adolescents kept under EE, but not those in SC, exhibited CPP. Among SC, but not among EE, adolescents, BDNF levels were significantly lower in those treated with ethanol than in those given vehicle. These results indicate that, compared to adults, adolescent exhibited reduced sensitivity to ethanol's rewarding effects, yet the youth but not the adults exhibited sensitivity to the promoting effect of EE upon CPP by ethanol. Ethanol significantly reduced BDNF levels in adolescents reared under standard housing conditions, but not in adult mice nor in adolescents given EE housing conditions. The present results add to the plethora of adolescent-specific responses to ethanol or to environmental stimuli that may put the youth at risk for escalation of ethanol intake.

Ethanol Sensitization during Adolescence or Adulthood Induces Different Patterns of Ethanol Consumption without Affecting Ethanol Metabolism.

In previous study, we demonstrated that ethanol preexposure may increase ethanol consumption in both adolescent and adult mice, in a two-bottle choice model. We now questioned if ethanol exposure during adolescence results in changes of consumption pattern using a three-bottle choice procedure, considering drinking-in-the-dark and alcohol deprivation effect as strategies for ethanol consumption escalation. We also analyzed aldehyde dehydrogenase (ALDH) activity as a measurement of ethanol metabolism. Adolescent and adult Swiss mice were treated with saline (SAL) or 2.0 g/kg ethanol (EtOH) during 15 days (groups: Adolescent-SAL, Adolescent-EtOH, Adult-SAL and Adult-EtOH). Five days after the last injection, mice were exposed to the three-bottle choice protocol using sucrose fading procedure (4% + sucrose vs. 8%-15% ethanol + sucrose vs. water + sucrose) for 2 h during the dark phase. Sucrose was faded out from 8% to 0%. The protocol was composed of a 6-week acquisition period, followed by four withdrawals and reexposures. Both adolescent and adult mice exhibited ethanol behavioral sensitization, although the magnitude of sensitization in adolescents was lower than in adults. Adolescent-EtOH displayed an escalation of 4% ethanol consumption during acquisition that was not observed in Adult-EtOH. Moreover, Adult-EtOH consumed less 4% ethanol throughout all the experiment and less 15% ethanol in the last reexposure period than Adolescent-EtOH. ALDH activity varied with age, in which older mice showed higher ALDH than younger ones. Ethanol pretreatment or the pattern of consumption did not have influence on ALDH activity. Our data suggest that ethanol pretreatment during adolescence but not adulthood may influence the pattern of ethanol consumption toward an escalation in ethanol consumption at low dose, without exerting an impact on ALDH activity.

Necroptosis drives motor neuron death in models of both sporadic and familial ALS.

Most cases of neurodegenerative diseases are sporadic, hindering the use of genetic mouse models to analyze disease mechanisms. Focusing on the motor neuron (MN) disease amyotrophic lateral sclerosis (ALS), we therefore devised a fully humanized coculture model composed of human adult primary sporadic ALS (sALS) astrocytes and human embryonic stem-cell-derived MNs. The model reproduces the cardinal features of human ALS: sALS astrocytes, but not those from control patients, trigger selective death of MNs. The mechanisms underlying this non-cell-autonomous toxicity were investigated in both astrocytes and MNs. Although causal in familial ALS (fALS), SOD1 does not contribute to the toxicity of sALS astrocytes. Death of MNs triggered by either sALS or fALS astrocytes occurs through necroptosis, a form of programmed necrosis involving receptor-interacting protein 1 and the mixed lineage kinase domain-like protein. The necroptotic pathway therefore constitutes a potential therapeutic target for this incurable disease.