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Stress affects the brain and alters its neuroarchitecture and function; these changes can be severe and lead to psychiatric disorders. Recent evidence suggests that astrocytes and microglia play an essential role in the stress response by contributing to the maintenance of cerebral homeostasis. These cells respond rapidly to all stimuli that reach the brain, including stressors. Here, we used a recently validated rodent model of post-traumatic stress disorder in which rats can be categorized as resilient or vulnerable after acute inescapable footshock stress. We then investigated the functional, molecular, and morphological determinants of stress resilience and vulnerability in the prefrontal cortex, focusing on glial and neuronal cells. In addition, we examined the effects of a single subanesthetic dose of ketamine, a fast-acting antidepressant recently approved for the treatment of resistant depression and proposed for other stress-related psychiatric disorders. The present results suggest a prompt glial cell response and activation of the NF-kappa B pathway after acute stress, leading to an increase in specific cytokines such as IL-18 and TNF-alpha. This response persists in vulnerable individuals and is accompanied by a significant change in the levels of critical glial proteins such as S100B, CD11b, and CX43, brain trophic factors such as BDNF and FGF2, and proteins related to dendritic arborization and synaptic architecture such as MAP2 and PSD95. Administration of ketamine 24 h after the acute stress event rescued many of the changes observed in vulnerable rats, possibly contributing to support brain homeostasis. Overall, our results suggest that pivotal events, including reactive astrogliosis, changes in brain trophic factors, and neuronal damage are critical determinants of vulnerability to acute traumatic stress and confirm the therapeutic effect of acute ketamine against the development of stress-related psychiatric disorders.

We previously described an approach called RealSeqS to evaluate aneuploidy in plasma cell-free DNA through the amplification of ~350,000 repeated elements with a single primer. We hypothesized that an unbiased evaluation of the large amount of sequencing data obtained with RealSeqS might reveal other differences between plasma samples from patients with and without cancer. This hypothesis was tested through the development of a machine learning approach called Alu Profile Learning Using Sequencing (A-PLUS) and its application to 7615 samples from 5178 individuals, 2073 with solid cancer and the remainder without cancer. Samples from patients with cancer and controls were prespecified into four cohorts used for model training, analyte integration, and threshold determination, validation, and reproducibility. A-PLUS alone provided a sensitivity of 40.5% across 11 different cancer types in the validation cohort, at a specificity of 98.5%. Combining A-PLUS with aneuploidy and eight common protein biomarkers detected 51% of the cancers at 98.9% specificity. We found that part of the power of A-PLUS could be ascribed to a single feature-the global reduction of AluS subfamily elements in the circulating DNA of patients with solid cancer. We confirmed this reduction through the analysis of another independent dataset obtained with a different approach (whole-genome sequencing). The evaluation of Alu elements may therefore have the potential to enhance the performance of several methods designed for the earlier detection of cancer.

Alzheimer’s disease (AD) is the most common age-related neurodegenerative disease characterized by memory loss and cognitive impairment. The causes of the disease are not well understood, as it involves a complex interaction between genetic, environmental, and epigenetic factors. SAMP8 mice have been proposed as a model for studying late-onset AD, since they show age-related learning and memory deficits as well as several features of AD pathogenesis. Epigenetic changes have been described in SAMP8 mice, although sex differences have never been evaluated. Here we used western blot and qPCR analyses to investigate whether epigenetic markers are differentially altered in the dorsal hippocampus, a region important for the regulation of learning and memory, of 9-month-old male and female SAMP8 mice. We found that H3Ac was selectively reduced in male SAMP8 mice compared to male SAMR1 control mice, but not in female mice, whereas H3K27me3 was reduced overall in SAMP8 mice. Moreover, the levels of HDAC2 and JmjD3 were increased, whereas the levels of HDAC4 and Dnmt3a were reduced in SAMP8 mice compared to SAMR1. In addition, levels of HDAC1 were reduced, whereas Utx and Jmjd3 were selectively increased in females compared to males. Although our results are preliminary, they suggest that epigenetic mechanisms in the dorsal hippocampus are differentially regulated in male and female SAMP8 mice.

Stress is a primary risk factor in the onset of neuropsychiatric disorders, including major depressive disorder (MDD). We have previously used the chronic mild stress (CMS) model of depression in male rats to show that CMS induces morphological, functional, and molecular changes in the hippocampus of vulnerable animals, the majority of which were recovered using acute subanesthetic ketamine in just 24 h. Here, we focused our attention on the medial prefrontal cortex (mPFC), a brain area regulating emotional and cognitive functions, and asked whether vulnerability/resilience to CMS and ketamine antidepressant effects were associated with molecular and functional changes in the mPFC of rats. We found that most alterations induced by CMS in the mPFC were selectively observed in stress-vulnerable animals and were rescued by acute subanesthetic ketamine, while others were found only in resilient animals or were induced by ketamine treatment. Importantly, only a few of these modifications were also previously demonstrated in the hippocampus, while most are specific to mPFC. Overall, our results suggest that acute antidepressant ketamine rescues brain-area-specific glutamatergic changes induced by chronic stress.

Frailty is a geriatric syndrome characterized by age-related decline in physiological reserves and functions in multiple organ systems, including the musculoskeletal, neuroendocrine/metabolic, and immune systems. Animal models are essential to study the biological basis of aging and potential ways to delay the onset of age-related phenotypes. Unfortunately, validated animal models of frailty are still lacking in preclinical research. The senescence-accelerated prone-8 (SAMP8) mouse strain exhibits early cognitive loss that mimics the deterioration of learning and memory in the elderly and is widely used as a model of aging and neurodegenerative diseases. Here, we examined the frailty phenotype, which includes body weight, strength, endurance, activity, and slow walking speed, in male and female SAMP8 and senescence-accelerated mouse resistant (SAMR1) mice at 6- and 9-months of age. We found that the prevalence of frailty was higher in SAMP8 mice compared with SAMR1 mice, regardless of sex. The overall percentage of prefrail and frail mice was similar in male and female SAMP8 mice, although the percentage of frail mice was slightly higher in males than in females. In addition, we found sex- and frailty-specific changes in selected miRNAs blood levels. In particular, the levels of miR-34a-5p and miR-331-3p were higher in both prefrail and frail mice, whereas miR-26b-5p was increased only in frail mice compared with robust mice. Finally, levels of miR-331-3p were also increased in whole blood from a small group of frail patients. Overall, these results suggest that SAMP8 mice may be a useful mouse model for identifying potential biomarkers and studying biological mechanisms of frailty.

Stress is a major risk factor for psychiatric disorders. During and after exposure to stressors, the stress response may have pro- or maladaptive consequences, depending on several factors related to the individual response and nature of the stressor. However, the mechanisms mediating the long-term effects of exposure to stress, which may ultimately lead to the development of stress-related disorders, are still largely unknown. Epigenetic mechanisms have been shown to mediate the effects of the environment on brain gene expression and behavior. MicroRNAs, small non-coding RNAs estimated to control the expression of about 60% of all genes by post-transcriptional regulation, are a fundamental epigenetic mechanism. Many microRNAs are expressed in the brain, where they work as fine-tuners of gene expression, with a key role in the regulation of homeostatic balance, and a likely influence on pro- or maladaptive brain changes. Here we have selected a number of microRNAs, which have been strongly implicated as mediators of the effects of stress in the brain and in the development of stress-related psychiatric disorders. For all of them recent evidence is reported, obtained from rodent stress models, manipulation of microRNAs levels with related behavioral changes, and clinical studies of stress-related psychiatric disorders. Moreover, we have performed a bioinformatic analysis of the predicted brain-expressed target genes of the microRNAs discussed, and found a central role for mechanisms involved in the regulation of synaptic function. The complex regulatory role of microRNAs has suggested their use as biomarkers for diagnosis and treatment response, as well as possible therapeutic drugs. While, microRNA-based diagnostics have registered advancements, particularly in oncology and other fields, and many biotech companies have launched miRNA therapeutics in their development pipeline, the development of microRNA-based tests and drugs for brain disorders is comparatively slo

Traumatic stress is the main environmental risk factor for the development of psychiatric disorders. We have previously shown that acute footshock (FS) stress in male rats induces rapid and long-lasting functional and structural changes in the prefrontal cortex (PFC), which are partly reversed by acute subanesthetic ketamine. Here, we asked if acute FS may also induce any changes in glutamatergic synaptic plasticity in the PFC 24 h after stress exposure and whether ketamine administration 6 h after stress may have any effect. We found that the induction of long-term potentiation (LTP) in PFC slices of both control and FS animals is dependent on dopamine and that dopamine-dependent LTP is reduced by ketamine. We also found selective changes in ionotropic glutamate receptor subunit expression, phosphorylation, and localization at synaptic membranes induced by both acute stress and ketamine. Although more studies are needed to understand the effects of acute stress and ketamine on PFC glutamatergic plasticity, this first report suggests a restoring effect of acute ketamine, supporting the potential benefit of ketamine in limiting the impact of acute traumatic stress.

Stress is a primary risk factor for psychiatric disorders such as Major Depressive Disorder (MDD) and Post Traumatic Stress Disorder (PTSD). The response to stress involves the regulation of transcriptional programs, which is supposed to play a role in coping with stress. To evaluate transcriptional processes implemented after exposure to unavoidable traumatic stress, we applied microarray expression analysis to the PFC of rats exposed to acute footshock (FS) stress that were sacrificed immediately after the 40 min session or 2 h or 24 h after. While no substantial changes were observed at the single gene level immediately after the stress session, gene set enrichment analysis showed alterations in neuronal pathways associated with glia development, glia-neuron networking, and synaptic function. Furthermore, we found alterations in the expression of gene sets regulated by specific transcription factors that could represent master regulators of the acute stress response. Of note, these pathways and transcriptional programs are activated during the early stress response (immediately after FS) and are already turned off after 2 h-while at 24 h, the transcriptional profile is largely unaffected. Overall, our analysis provided a transcriptional landscape of the early changes triggered by acute unavoidable FS stress in the PFC of rats, suggesting that the transcriptional wave is fast and mild, but probably enough to activate a cellular response to acute stress.

Stress represents a main risk factor for psychiatric disorders. Whereas it is known that even a single trauma may induce psychiatric disorders in humans, the mechanisms of vulnerability to acute stressors have been little investigated. In this study, we generated a new animal model of resilience/vulnerability to acute footshock (FS) stress in rats and analyzed early functional, molecular, and morphological determinants of stress vulnerability at tripartite glutamate synapses in the prefrontal cortex (PFC). We found that adult male rats subjected to FS can be deemed resilient (FS-R) or vulnerable (FS-V), based on their anhedonic phenotype 24 h after stress exposure, and that these two populations are phenotypically distinguishable up to two weeks afterwards. Basal presynaptic glutamate release was increased in the PFC of FS-V rats, while depolarization-evoked glutamate release and synapsin I phosphorylation at Ser9 were increased in both FS-R and FS-V. In FS-R and FS-V rats the synaptic expression of GluN2A and apical dendritic length of prelimbic PFC layers II–III pyramidal neurons were decreased, while BDNF expression was selectively reduced in FS-V. Depolarization-evoked (carrier-mediated) glutamate release from astroglia perisynaptic processes (gliosomes) was selectively increased in the PFC of FS-V rats, while GLT1 and xCt levels were higher and GS expression reduced in purified PFC gliosomes from FS-R. Overall, we show for the first time that the application of the sucrose intake test to rats exposed to acute FS led to the generation of a novel animal model of resilience/vulnerability to acute stress, which we used to identify early determinants of maladaptive response related to behavioral vulnerability to stress.

Stress is a key risk factor in the onset of neuropsychiatric disorders. The study of the mechanisms underlying stress response is important to understand the etiopathogenetic mechanisms and identify new putative therapeutic targets. In this context, microRNAs (miRNAs) have emerged as key regulators of the complex patterns of gene/protein expression changes in the brain, where they have a crucial role in the regulation of neuroplasticity, neurogenesis, and neuronal differentiation. Among them, miR-135a-5p has been associated with stress response, synaptic plasticity, and the antidepressant effect in different brain areas. Here, we used acute unavoidable foot-shock stress (FS) and chronic mild stress (CMS) on male rats to study whether miR-135a-5p was involved in stress-induced changes in the prefrontal cortex (PFC). Both acute and chronic stress decreased miR-135a-5p levels in the PFC, although after CMS the reduction was induced only in animals vulnerable to CMS, according to a sucrose preference test. MiR-135a-5p downregulation in the primary neurons reduced dendritic spine density, while its overexpression exerted the opposite effect. Two bioinformatically predicted target genes, Kif5c and Cplx1/2, were increased in FS rats 24 h after stress. Altogether, we found that miR-135a-5p might play a role in stress response in PFC involving synaptic mechanisms.

Stress represents a major risk factor for psychiatric disorders, including post-traumatic stress disorder (PTSD). Recently, we dissected the destabilizing effects of acute stress on the excitatory glutamate system in the prefrontal cortex (PFC). Here, we assessed the effects of single subanesthetic administration of ketamine (10 mg/kg) on glutamate transmission and dendritic arborization in the PFC of footshock (FS)-stressed rats, along with changes in depressive, anxious, and fear extinction behaviors. We found that ketamine, while inducing a mild increase of glutamate release in the PFC of naïve rats, blocked the acute stress-induced enhancement of glutamate release when administered 24 or 72 h before or 6 h after FS. Accordingly, the treatment with ketamine 6 h after FS also reduced the stress-dependent increase of spontaneous excitatory postsynaptic current (sEPSC) amplitude in prelimbic (PL)-PFC. At the same time, ketamine injection 6 h after FS was found to rescue apical dendritic retraction of pyramidal neurons induced by acute stress in PL-PFC and facilitated contextual fear extinction. These results show rapid effects of ketamine in animals subjected to acute FS, in line with previous studies suggesting a therapeutic action of the drug in PTSD models. Our data are consistent with a mechanism of ketamine involving re-establishment of synaptic homeostasis, through restoration of glutamate release, and structural remodeling of dendrites.

Frailty is an aging-related pathology, defined as a state of increased vulnerability to stressors, leading to a limited capacity to meet homeostatic demands. Extracellular microRNAs (miRNAs) were proposed as potential biomarkers of various disease conditions, including age-related pathologies. The primary objective of this study was to identify blood miRNAs that could serve as potential biomarkers and candidate mechanisms of frailty. Using the Fried index, we enrolled 22 robust and 19 frail subjects. Blood and urine samples were analysed for several biochemical parameters. We observed that sTNF-R was robustly upregulated in the frail group, indicating the presence of an inflammatory state. Further, by RNA-seq, we profiled 2654 mature miRNAs in the whole blood of the two groups. Expression levels of selected differentially expressed miRNAs were validated by qPCR, and target prediction analyses were performed for the dysregulated miRNAs. We identified 2 miRNAs able to significantly differentiate frail patients from robust subjects. Both miR-101-3p and miR-142-5p were found to be downregulated in the frail vs. robust group. Finally, using bioinformatics targets prediction tools, we explored the potential molecular mechanisms and cellular pathways regulated by the two miRNAs and potentially involved in frailty.

Stressful life events are considered major risk factors for the development of several psychiatric disorders, though people differentially cope with stress. The reasons for this are still largely unknown but could be accounted for by individual genetic variants, previous life events, or the kind of stressors. The human brain-derived neurotrophic factor (BDNF) Val66Met variant, which was found to impair intracellular trafficking and activity-dependent secretion of BDNF, has been associated with increased susceptibility to develop several neuropsychiatric disorders, although there is still some controversial evidence. On the other hand, acute stress has been consistently demonstrated to promote the release of glutamate in cortico-limbic regions and altered glutamatergic transmission has been reported in psychiatric disorders. However, it is not known if the BDNF Val66Met single-nucleotide polymorphism (SNP) affects the stress-induced presynaptic glutamate release. In this study, we exposed adult male BDNFVal/Val and BDNFVal/Met knock-in mice to 30 min of acute restraint stress. Plasma corticosterone levels, glutamate release, protein, and gene expression in the hippocampus were analyzed immediately after the end of the stress session. Acute restraint stress similarly increased plasma corticosterone levels and nuclear glucocorticoid receptor levels and phosphorylation in both BDNFVal/Val and BDNFVal/Met mice. However, acute restraint stress induced higher increases in hippocampal presynaptic release of glutamate, phosphorylation of cAMP-response element binding protein (CREB), and levels of the immediate early gene c-fos of BDNFVal/Met compared to BFNFVal/Val mice. Moreover, acute restraint stress selectively increased phosphorylation levels of synapsin I at Ser9 and at Ser603 in BDNFVal/Val and BDNFVal/Met mice, respectively. In conclusion, we report here that the BDNF Val66Met SNP knock-in mice display an altered response to acute restraint stress in terms of hippoca

Posttraumatic Stress Disorder (PTSD) is a chronic mental illness with limited options for treatment, characterized by intrusive memory of trauma, avoidance, hyperarousal, emotional numbing, and anhedonia. PTSD is often triggered by exposure to a single traumatic experience, with high prevalence among war veterans. PTSD, together with other neuropsychiatric disorders, involves long-term changes in the structure and function of brain areas, synaptic disconnection, and changes in large-scale brain networks. Trauma-focused psychotherapy is considered first-line treatment, with greater and more persistent efficacy than pharmacotherapeutic approaches. Pharmacological treatments include SSRI, particularly paroxetine and sertraline, and other traditional antidepressants. The effect size of efficacy for these drugs is often small, with high treatment resistance in certain populations. In recent years a major shift in the conceptual framework of neuropsychiatric disorders has occurred, from the monoamine hypothesis to a neuroplasticity hypothesis, in which the glutamate system is conceived as a primary mediator of pathology and a straight target for antidepressant drugs. Novel potential treatment options have emerged in recent years, in particular several modulators of the glutamate system, including ketamine, riluzole, D-cycloserine, N-acetylcysteine. Ketamine, the prototypical rapid-acting antidepressant, has received much attention for its complex mechanism of action (including clinical trials), and has been proposed as a prophylactic agent against the onset of PTSD after exposure to traumatic stress. This chapter will explore new pharmacological approaches to the therapy of PTSD, based on the modulation of the glutamate system.

The analysis of cell-free DNA (cfDNA) from plasma offers great promise for the earlier detection of cancer. At present, changes in DNA sequence, methylation, or copy number are the most sensitive ways to detect the presence of cancer. To further increase the sensitivity of such assays with limited amounts of sample, it would be useful to be able to evaluate the same template molecules for all these changes. Here, we report an approach, called MethylSaferSeqS, that achieves this goal, and can be applied to any standard library preparation method suitable for massively parallel sequencing. The innovative step was to copy both strands of each DNA-barcoded molecule with a primer that allows the subsequent separation of the original strands (retaining their 5-methylcytosine residues) from the copied strands (in which the 5-methylcytosine residues are replaced with unmodified cytosine residues). The epigenetic and genetic alterations present in the DNA molecules can then be obtained from the original and copied strands, respectively. We applied this approach to plasma from 265 individuals, including 198 with cancers of the pancreas, ovary, lung, and colon, and found the expected patterns of mutations, copy number alterations, and methylation. Furthermore, we could determine which original template DNA molecules were methylated and/or mutated. MethylSaferSeqS should be useful for addressing a variety of questions relating genetics and epigenetics.

Frailty is an aging related condition, which has been defined as a state of enhanced vulnerability to stressors, leading to a limited capacity to meet homeostatic demands. Cognitive impairment is also frequent in older people, often accompanying frailty. Age is the main independent risk factor for both frailty and cognitive impairment, and compelling evidence suggests that similar age-associated mechanisms could underlie both clinical conditions. Accordingly, it has been suggested that frailty and cognitive impairment share common pathways, and some authors proposed “cognitive frailty” as a single complex phenotype. Nevertheless, so far, no clear common underlying pathways have been discovered for both conditions. microRNAs (miRNAs) have emerged as key fine-tuning regulators in most physiological processes, as well as pathological conditions. Importantly, miRNAs have been proposed as both peripheral biomarkers and potential molecular factors involved in physiological and pathological aging. In this review, we discuss the evidence linking changes of selected miRNAs expression with frailty and cognitive impairment. Overall, miR-92a-5p and miR-532-5p, as well as other miRNAs implicated in pathological aging, should be investigated as potential biomarkers (and putative molecular effectors) of cognitive frailty.

Anxiety disorders are common mental health diseases affecting up to 7% of people around the world. Stress is considered one of the major environmental risk factors to promote anxiety disorders through mechanisms involving epigenetic changes. Moreover, alteration in redox balance and increased reactive oxygen species (ROS) production have been detected in anxiety patients and in stressed-animal models of anxiety. Here we tested if the administration of apocynin, a natural origin antioxidant, may prevent the anxiety-like phenotype and reduction of histone acetylation induced by a subchronic forced swimming stress (FSS) paradigm. We found that apocynin prevented the enhanced latency time in the novelty-suppressed feeding test, and the production of malondialdehyde induced by FSS. Moreover, apocynin was able to block the upregulation of p47phox, a key subunit of the NADPH oxidase complex. Finally, apocynin prevented the rise of hippocampal Hdac1, Hdac4 and Hdac5, and the reduction of histone-3 acetylation levels promoted by FSS exposure. In conclusion, our results provide evidence that apocynin reduces the deleterious effect of stress and suggests that oxidative stress may regulate epigenetic mechanisms.

Stress plays a crucial role in the pathogenesis of psychiatric disorders and affects neuronal plasticity in different brain regions. We have previously found that acute foot-shock (FS) stress elicits fast and long-lasting functional and morphological remodeling of excitatory neurons in the prefrontal cortex (PFC), which were partly prevented by the pretreatment with antidepressants. Here we investigated, whether acute stress and pretreatment with desipramine (DMI) interfere in hippocampal dendritic remodeling. Male Sprague-Dawley rats were subjected to acute FS-stress, followed by measurement of time-dependent (1, 7 and 14 days) structural plasticity (dendritic arborization, spine number and morphology) in hippocampal CA1 pyramidal neurons and expression patterns of molecular markers implicated in neuronal plasticity. We found that acute stress significantly decreased spine number, dendritic length, and altered spine morphometric parameters at all time points evaluated after stress. This was paralleled by changes in the gene expression of Spinophilin and Cdc42, and protein expression of homer1. Pretreatment with DMI prevented the stress-induced dendritic atrophy and spine loss 14 days after acute FS. However, DMI treatment without stress differentially affected the expression patterns of spine-related genes and proteins. In conclusion, acute FS-stress and pretreatment with DMI significantly changed dendritic morphology, including number and morphology of spines, and the length of dendrites in hippocampal CA1 pyramidal cells as early as 1 day, and sustained up to 14 days after acute FS. The findings were paralleled by changes in gene and protein expression of actin binding and cytoskeletal proteins, Rho GTPases, and postsynaptic scaffolding proteins.

Depression is a debilitating mental disease, characterized by persistent low mood and anhedonia. Stress represents a major environmental risk factor for depression; the complex interaction of stress with genetic factors results in different individual vulnerability or resilience to the disorder. Dysfunctions of the glutamate system have a primary role in depression. Clinical neuroimaging studies have consistently reported alterations in volume and connectivity of cortico-limbic areas, where glutamate neurons and synapses predominate. This is confirmed by preclinical studies in rodents, showing that repeated stress induces morphological and functional maladaptive changes in the same brain regions altered in humans. Confirming the key role of glutamatergic transmission in depression, compelling evidence has shown that the non-competitive NMDA receptor antagonist, ketamine, induces, at sub-anesthetic dose, rapid and sustained antidepressant response in both humans and rodents. We show here that the Chronic Mild Stress model of depression induces, only in stress-vulnerable rats, depressed-like anhedonic behavior, together with impairment of glutamate/GABA presynaptic release, BDNF mRNA trafficking in dendrites and dendritic morphology in hippocampus. Moreover, we show that a single administration of ketamine restores, in 24 h, normal behavior and most of the cellular/molecular maladaptive changes in vulnerable rats. Interestingly, ketamine treatment did not restore BDNF mRNA levels reduced by chronic stress but rescued dendritic trafficking of BDNF mRNA. The present results are consistent with a mechanism of ketamine involving rapid restoration of synaptic homeostasis, through re-equilibration of glutamate/GABA release and dendritic BDNF for synaptic translation and reversal of synaptic and circuitry impairment.

When administered as a single subanesthetic dose, the n-methyl-d-aspartate (NMDA) receptor antagonist, ketamine, produces rapid (within hours) and relatively sustained antidepressant actions even in treatment-resistant patients. Preclinical studies have shown that ketamine increases dendritic spine density and synaptic proteins in brain areas critical for the actions of antidepressants, yet the temporal relationship between structural changes and the onset of antidepressant action remains poorly understood. In this study, we examined the effects of a single dose of S-ketamine (15 mg/kg) on dendritic length, dendritic arborization, spine density, and spine morphology in the Flinders Sensitive and Flinders Resistant Line (FSL/FRL) rat model of depression. We found that already 1 h after injection with ketamine, apical dendritic spine deficits in CA1 pyramidal neurons of FSL rats were completely restored. Notably, the observed increase in spine density was attributable to regulation of both mushroom and long-thin spines. In contrast, ketamine had no effect on dendritic spine density in FRL rats. On the molecular level, ketamine normalized elevated levels of phospho-cofilin and the NMDA receptor subunits GluN2A and GluN2B and reversed homer3 deficiency in hippocampal synaptosomes of FSL rats. Taken together, our data suggest that rapid formation of new spines may provide an important structural substrate during the initial phase of ketamine’s antidepressant action.

Converging clinical and preclinical evidence demonstrates that depressive phenotypes are associated with synaptic dysfunction and dendritic simplification in cortico-limbic glutamatergic areas. On the other hand, the rapid antidepressant effect of acute ketamine is consistently reported to occur together with the rescue of dendritic atrophy and reduction of spine number induced by chronic stress in the hippocampus and prefrontal cortex of animal models of depression. Nevertheless, the molecular mechanisms underlying these morphological alterations remain largely unknown. Here, we found that miR-9-5p levels were selectively reduced in the hippocampus of rats vulnerable to Chronic Mild Stress (CMS), while acute subanesthetic ketamine restored its levels to basal condition in just 24h; miR-9-5p expression inversely correlated with the anhedonic phenotype. A decrease of miR-9-5p was reproduced in an in vitro model of stress, based on primary hippocampal neurons incubated with the stress hormone corticosterone. In both CMS animals and primary neurons, decreased miR-9-5p levels were associated with dendritic simplification, while treatment with ketamine completely rescued the changes. In vitro modulation of miR-9-5p expression showed a direct role of miR-9-5p in regulating dendritic length and spine density in mature primary hippocampal neurons. Among the putative target genes tested, Rest and Sirt1 were validated as biological targets in primary neuronal cultures. Moreover, in line with miR-9-5p changes, REST protein expression levels were remarkably increased in both CMS vulnerable animals and corticosterone-treated neurons, while ketamine completely abolished this alteration. Finally, the shortening of dendritic length in corticosterone-treated neurons was shown to be partly rescued by miR-9-5p overexpression and dependent on REST protein expression. Overall, our data unveiled the functional role of miR-9-5p in the remodeling of dendritic arbor induced by stress/cortic

Identification and quantification of low-frequency mutations remain challenging despite improvements in the baseline error rate of next-generation sequencing technologies. Here, we describe a method, termed SaferSeqS, that addresses these challenges by (1) efficiently introducing identical molecular barcodes in the Watson and Crick strands of template molecules and (2) enriching target sequences with strand-specific PCR. The method achieves high sensitivity and specificity and detects variants at frequencies below 1 in 100,000 DNA template molecules with a background mutation rate of <5 × 10-7 mutants per base pair (bp). We demonstrate that it can evaluate mutations in a single amplicon or simultaneously in multiple amplicons, assess limited quantities of cell-free DNA with high recovery of both strands and reduce the error rate of existing PCR-based molecular barcoding approaches by >100-fold.

Studies in multiple solid tumor types have demonstrated the prognostic significance of ctDNA analysis after curative intent surgery. A combined analysis of data across completed studies could further our understanding of circulating tumor DNA (ctDNA) as a prognostic marker and inform future trial design. We combined individual patient data from three independent cohort studies of nonmetastatic colorectal cancer (CRC). Plasma samples were collected 4 to 10 weeks after surgery. Mutations in ctDNA were assayed using a massively parallel sequencing technique called SafeSeqS. We analyzed 485 CRC patients (230 Stage II colon, 96 Stage III colon, and 159 locally advanced rectum). ctDNA was detected after surgery in 59 (12%) patients overall (11.0%, 12.5% and 13.8% for samples taken at 4-6, 6-8 and 8-10 weeks; P = .740). ctDNA detection was associated with poorer 5-year recurrence-free (38.6% vs 85.5%; P < .001) and overall survival (64.6% vs 89.4%; P < .001). The predictive accuracy of postsurgery ctDNA for recurrence was higher than that of individual clinicopathologic risk features. Recurrence risk increased exponentially with increasing ctDNA mutant allele frequency (MAF) (hazard ratio, 1.2, 2.5 and 5.8 for MAF of 0.1%, 0.5% and 1%). Postsurgery ctDNA was detected in 3 of 20 (15%) patients with locoregional and 27 of 60 (45%) with distant recurrence (P = .018). This analysis demonstrates a consistent long-term impact of ctDNA as a prognostic marker across nonmetastatic CRC, where ctDNA outperforms other clinicopathologic risk factors and MAF further stratifies recurrence risk. ctDNA is a better predictor of distant vs locoregional recurrence.

Stress is a major risk factor in the onset of neuropsychiatric diseases including mood disorders. Stress-based animal models of depression have shown dendritic atrophy and spine remodeling in the hippocampus (HPC), suggesting that stress-induced maladaptive changes have a primary role in the psychopathology. Recent studies showed that a single sub-anesthetic dose of ketamine (KET) exerts a fast and sustained antidepressant effect, accompanied by a rapid reverse of synaptic deficits. However, the molecular mechanisms that underlie the functional and morphological changes induced by stress and KET are still largely unknown. MicroRNA (miRNAs) have emerged as regulators of complex patterns of gene/protein expression changes in the brain and recent studies showed their involvement in the pathophysiology of mood disorders and in the action of psychotropic drugs. Their peculiar ability to fine-tune the expression of hundreds of genes makes them potential candidates as fast mediators of stress-response and KET action. Here we used the Chronic Mild Stress (CMS) animal model of depression and in vitro primary HPC cultures to study whether mechanisms of stress response and KET fast antidepressant action involved changes in dendrite remodeling of HPC pyramidal neurons, together with alteration in the expression of miRNAs known to play a role in neuroplasticity and stress response. Morphological analysis of CA3 pyramidal neurons showed, selectively in animals vulnerable to CMS, a reduction in total length and branching of apical dendrites; KET completely restored these changes. At a molecular level, the expression of miR-9-5p, a brain-enriched miRNA previously associated with neuronal morphology regulation, was selectively decreased in vulnerable animals, while KET recovered the reduction. Further, treatment of primary HPC neurons with corticosterone reduced both dendritic length and miR-9-5p levels; KET fully reversed the changes. By modulating miR-9-5p expression in primary HP

The Negr1 gene has been significantly associated with major depression in genetic studies. Negr1 encodes for a cell adhesion molecule cleaved by the protease Adam10, thus activating Fgfr2 and promoting neuronal spine plasticity. We investigated whether antidepressants modulate the expression of genes belonging to Negr1-Fgfr2 pathway in Flinders sensitive line (FSL) rats, in a corticosterone-treated mouse model of depression, and in mouse primary neurons. Negr1 and Adam10 were the genes mostly affected by antidepressant treatment, and in opposite directions. Negr1 was down-regulated by escitalopram in the hypothalamus of FSL rats, by fluoxetine in the hippocampal dentate gyrus of corticosterone-treated mice, and by nortriptyline in hippocampal primary neurons. Adam10 mRNA was increased by nortriptyline administration in the hypothalamus, by escitalopram in the hippocampus of FSL rats, and by fluoxetine in mouse dorsal dentate gyrus. Similarly, nortriptyline increased Adam10 expression in hippocampal cultures. Fgfr2 expression was increased by nortriptyline in the hypothalamus of FSL rats and in hippocampal neurons. Lsamp, another IgLON family protein, increased in mouse dentate gyrus after fluoxetine treatment. These findings suggest that Negr1-Fgfr2 pathway plays a role in the modulation of synaptic plasticity induced by antidepressant treatment to promote therapeutic efficacy by rearranging connectivity in corticolimbic circuits impaired in depression.

Neurobiological underpinnings of treatment-resistant depression, a debilitating condition associated with significant functional impairment, have not been elucidated. Consequently, the aim of this study was to use animal models of response and resistance to antidepressant treatment, in an attempt to identify differences in associated transcriptional responses. Flinders Sensitive Line rats were subjected to maternal separation (MS) and chronically treated with Escitalopram or Nortriptyline. Antidepressants reduced immobility time in the forced swim test in non-MS rats, while lack of antidepressant behavioural response was observed in MS animals. We developed a novel bioinformatic algorithm that enabled identification of transcriptional signatures in hippocampus and pre-frontal cortex that discriminate vehicle- and antidepressant-treated subjects in both MS and non-MS rats. Functional annotation analysis showed that in antidepressant-responder rats the most enriched pathways included IQGAPs activation, toll-like receptor trafficking, energy metabolism, and regulation of endopeptidase activity. The analysis of interacting proteins implicated synaptic vesicles and neurotransmitter release, ubiquitin regulation, cytoskeleton organisation and carbohydrate metabolism. In contrast, in treatment-resistant MS rats, main expression changes were revealed in ribosomal proteins, inflammatory responses, transcriptional/epigenetic regulation, and small GTPases. Susceptibility signature shared Rtn1, Zdhhc5, Igsf6, and Sim1 genes with the latest depression GWAS meta-analysis, while antidepressant resistance signature shared Ctnnd1, Rbms3, Atp1a3, and Pla2r1 genes. In conclusion, this study demonstrated that distinct transcriptional signatures are associated with behavioural response or non-response to antidepressant treatment. The identification of genes involved in antidepressant response will increase the comprehension of the neurobiological underpinnings of treatment-resistant dep