Somatic Regions Research Articles

Generation of contractile forces by three-dimensional bundled axonal tracts in micro-tissue engineered neural networks.

Axonal extension and retraction are ongoing processes that occur throughout all developmental stages of an organism. The ability of axons to produce mechanical forces internally and respond to externally generated forces is crucial for nervous system development, maintenance, and plasticity. Such axonal mechanobiological phenomena have typically been evaluated in vitro at a single-cell level, but these mechanisms have not been studied when axons are present in a bundled three-dimensional (3D) form like in native tissue. In an attempt to emulate native cortico-cortical interactions under in vitro conditions, we present our approach to utilize previously described micro-tissue engineered neural networks (micro-TENNs). Here, micro-TENNs were comprised of discrete populations of rat cortical neurons that were spanned by 3D bundled axonal tracts and physically integrated with each other. We found that these bundled axonal tracts inherently exhibited an ability to generate contractile forces as the microtissue matured. We therefore utilized this micro-TENN testbed to characterize the intrinsic contractile forces generated by the integrated axonal tracts in the absence of any external force. We found that contractile forces generated by bundled axons were dependent on microtubule stability. Moreover, these intra-axonal contractile forces could simultaneously generate tensile forces to induce so-called axonal "stretch-growth" in different axonal tracts within the same microtissue. The culmination of axonal contraction generally occurred with the fusion of both the neuronal somatic regions along the axonal tracts, therefore perhaps showing the innate tendency of cortical neurons to minimize their wiring distance, a phenomenon also perceived during brain morphogenesis. In future applications, this testbed may be used to investigate mechanisms of neuroanatomical development and those underlying certain neurodevelopmental disorders.

Structural and functional characterization of the IgSF21-neurexin2α complex and its related signaling pathways in the regulation of inhibitory synapse organization.

The prevailing model behind synapse development and specificity is that a multitude of adhesion molecules engage in transsynaptic interactions to induce pre- and postsynaptic assembly. How these extracellular interactions translate into intracellular signal transduction for synaptic assembly remains unclear. Here, we focus on a synapse organizing complex formed by immunoglobulin superfamily member 21 (IgSF21) and neurexin2α (Nrxn2α) that regulates GABAergic synapse development in the mouse brain. We reveal that the interaction between presynaptic Nrxn2α and postsynaptic IgSF21 is a high-affinity receptor-ligand interaction and identify a binding interface in the IgSF21-Nrxn2α complex. Despite being expressed in both dendritic and somatic regions, IgSF21 preferentially regulates dendritic GABAergic presynaptic differentiation whereas another canonical Nrxn ligand, neuroligin2 (Nlgn2), primarily regulates perisomatic presynaptic differentiation. To explore mechanisms that could underlie this compartment specificity, we targeted multiple signaling pathways pharmacologically while monitoring the synaptogenic activity of IgSF21 and Nlgn2. Interestingly, both IgSF21 and Nlgn2 require c-jun N-terminal kinase (JNK)-mediated signaling, whereas Nlgn2, but not IgSF21, additionally requires CaMKII and Src kinase activity. JNK inhibition diminished de novo presynaptic differentiation without affecting the maintenance of formed synapses. We further found that Nrxn2α knockout brains exhibit altered synaptic JNK activity in a sex-specific fashion, suggesting functional linkage between Nrxns and JNK. Thus, our study elucidates the structural and functional relationship of IgSF21 with Nrxn2α and distinct signaling pathways for IgSF21-Nrxn2α and Nlgn2-Nrxn synaptic organizing complexes in vitro. We therefore propose a revised hypothesis that Nrxns act as molecular hubs to specify synaptic properties not only through their multiple extracellular ligands but also through distinct intracellular signaling pathways of these ligands.

Lifetime Stressful Events Associated with Alzheimer's Pathologies, Neuroinflammation and Brain Structure in a Risk Enriched Cohort.

Along with the known effects of stress on brain structure and inflammatory processes, increasing evidence suggest a role of chronic stress in the pathogenesis of Alzheimer's disease (AD). We investigated the association of accumulated stressful life events (SLEs) with AD pathologies, neuroinflammation, and gray matter (GM) volume among cognitively unimpaired (CU) individuals at heightened risk of AD. This cross-sectional cohort study included 1,290 CU participants (aged 48-77) from the ALFA cohort with SLE, lumbar puncture (n = 393), and/or structural magnetic resonance imaging (n = 1,234) assessments. Using multiple regression analyses, we examined the associations of total SLEs with cerebrospinal fluid (1) phosphorylated (p)-tau181 and Aβ1-42/1-40 ratio, (2) interleukin 6 (IL-6), and (3) GM volumes voxel-wise. Further, we performed stratified and interaction analyses with sex, history of psychiatric disease, and evaluated SLEs during specific life periods. Within the whole sample, only childhood and midlife SLEs, but not total SLEs, were associated with AD pathophysiology and neuroinflammation. Among those with a history of psychiatric disease SLEs were associated with higher p-tau181 and IL-6. Participants with history of psychiatric disease and men, showed lower Aβ1-42/1-40 with higher SLEs. Participants with history of psychiatric disease and women showed reduced GM volumes in somatic regions and prefrontal and limbic regions, respectively. We did not find evidence supporting the association of total SLEs with AD, neuroinflammation, and atrophy pathways. Instead, the associations appear to be contingent on events occurring during early and midlife, sex and history of psychiatric disease. ANN NEUROL 2024;95:1058-1068.

γ-Aminobutyric acid transporter and GABAA receptor mechanisms in Slc6a1+/A288V and Slc6a1+/S295L mice associated with developmental and epileptic encephalopathies.

We have previously characterized the molecular mechanisms for variants in γ-aminobutyric acid transporter 1-encoding solute carrier family 6-member 1 (SLC6A1) in vitro and concluded that a partial or complete loss of γ-aminobutyric acid uptake due to impaired protein trafficking is the primary aetiology. Impairment of γ-aminobutyric acid transporter 1 function could cause compensatory changes in the expression of γ-aminobutyric acid receptors, which, in turn, modify disease pathophysiology and phenotype. Here we used different approaches including radioactive 3H γ-aminobutyric acid uptake in cells and synaptosomes, immunohistochemistry and confocal microscopy as well as brain slice surface protein biotinylation to characterize Slc6a1+/A288V and Slc6a1+/S295L mice, representative of a partial or a complete loss of function of SLC6A1 mutations, respectively. We employed the γ-aminobutyric acid transporter 1-specific inhibitor [3H]tiagabine binding and GABAA receptor subunit-specific radioligand binding to profile the γ-aminobutyric acid transporter 1 and GABAA receptor expression in major brain regions such as cortex, cerebellum, hippocampus and thalamus. We also determined the total and surface expression of γ-aminobutyric acid transporter 1, γ-aminobutyric acid transporter 3 and expression of GABAA receptor in the major brain regions in the knockin mice. We found that γ-aminobutyric acid transporter 1 protein was markedly reduced in cortex, hippocampus, thalamus and cerebellum in both mutant mouse lines. Consistent with the findings of reduced γ-aminobutyric acid uptake for both γ-aminobutyric acid transporter 1(A288V) and γ-aminobutyric acid transporter 1(S295L), both the total and the γ-aminobutyric acid transporter 1-mediated 3H γ-aminobutyric acid reuptake was reduced. We found that γ-aminobutyric acid transporter 3 is only abundantly expressed in the thalamus and there was no compensatory increase of γ-aminobutyric acid transporter 3 in either of the mutant mouse lines. γ-Aminobutyric acid transporter 1 was reduced in both somatic regions and nonsomatic regions in both mouse models, in which a ring-like structure was identified only in the Slc6a1+/A288V mouse, suggesting more γ-aminobutyric acid transporter 1 retention inside endoplasmic reticulum in the Slc6a1+/A288V mouse. The [3H]tiagabine binding was similar in both mouse models despite the difference in γ-aminobutyric acid uptake function and γ-aminobutyric acid transporter 1 protein expression for both mutations. There were no differences in GABAA receptor subtype expression, except for a small increase in the expression of α5 subunits of GABAA receptor in the hippocampus of Slc6a1S295L homozygous mice, suggesting a potential interaction between the expression of this GABAA receptor subtype and the mutant γ-aminobutyric acid transporter 1. The study provides the first comprehensive characterization of the SLC6A1 mutations in vivo in two representative mouse models. Because both γ-aminobutyric acid transporter 1 and GABAA receptors are targets for anti-seizure medications, the findings from this study can help guide tailored treatment options based on the expression and function of γ-aminobutyric acid transporter 1 and GABAA receptor in SLC6A1 mutation-mediated neurodevelopmental and epileptic encephalopathies.

Peripheral neural mechanism of visceral pain and acupoint sensitization in 2, 4, 6-trinitrobenzene sulfonic acid-induced colitis model mice.

To explore the neural mechanism of visceral pain and related somatic (acupoints) sensitization by using in vivo calcium imaging of dorsal root ganglia (DRG) neurons. Eight BALB/c mice were randomly divided into control and model groups, with 4 mice in each group. The colitis model was induced by colorectal perfusion of 2, 4, 6-trinitrobenzene sulfonic acid (TNBS) once daily for 7 days. Mice of the control group received colorectal perfusion of normal saline once daily for 7 days. The location and area of the somatic neurogenic inflammation (cutaneous exudation of Evans blue ［EB］) of the 2 groups of mice were observed after intravenous injection of EB. For pain behavioral tests, sixteen C57BL/6J mice were randomly divided into control and model groups, with 8 mice in each group, and a Von Frey filament was used to stimulate the referred somatic reactive regions in colitis mice, and the number of avoidance and paw withdraw reaction within 10 tests was recorded. For in vivo DRG calcium imaging tests, 24 Pirt-GCaMP6s transgenic mice were randomly and equally divided into control group and colitis model group. The responses of the neurons in L6 or L4 DRG to colorectal distension (CRD), lower back brushing, or mechanical stimulation at the hindpaw were observed using confocal fluorescence microscope. Compared with the control group, the area of EB exudation spot in the hindpaw and lower back regions was increased in the colitis model group (P<0.05), and the avoidance or paw withdraw numbers induced by Von Frey stimulation at the lower back and hindpaw were increased (P<0.01, P<0.05), indicating that colitis induced regional skin (acupoints) sensitization in the lower back and hindpaw regions. Compared with the control group, the percentage of L6 DRG neurons activated by 60 mm Hg CRD in the colitis model mice were apparently increased (P<0.01), the activated neurons mainly involved the medium-sized DRG neurons (P<0.01). In Pirt-GCaMP6s transgenic mice, following brushing the skin of the receptive field (lower back) of L6 DRG neurons, the fluorescence intensity of the brushing-activated DRG neurons and small, medium and large-sized neurons were significantly higher in the colitis model group than those in the control group (P<0.001, P<0.01, P<0.05). After brushing and clamping the skin of the right hindpaw (receptive field of L4 DRG neurons), the percentages of the activated L4 DRG neurons were obviously higher in the colitis model group than those in the control group (P<0.01, P<0.05), while there were no significant changes in the proportion of small, medium and large-sized neurons between the control and colitis model groups. Colitis may lead to body surface sensitization at the same and adjacent neuro-segments as well as to an increase of the number and activity of the responsive lumbar DRG neurons, among which the L6 DRG neurons at the same neuro-segment as the rectum colon showed an increase in the number of responders and intensity of calcium fluorescence signal while L4 DRG neurons at the level adjacent to the rectum colon showed an increase in the number of responders, suggesting that there may be different mechanisms of peripheral neural sensitization.

Abstract 6094: Longitudinal analysis of PARP inhibitor and platinum resistance in BRCA1/2m breast cancer using liquid biopsy

Abstract Background: Assessing how clinical resistance to PARP inhibitors develops has been challenging, due to the lack of coverage of potential resistance genes in sequencing panels and biopsies being subject to spatial heterogeneity. We studied the development of PARPi and/or platinum resistant disease using both tissue and a novel liquid biopsy assay, in patients treated for BRCA1/2-mutated metastatic breast cancer (BRCA1/2m mBC). Approach: A cohort of 35 mBC patients with germline or somatic BRCA1/2 mutations were identified as having developed PARPi or platinum resistant disease. Tumour biopsies were analysed by exome and transcriptome sequencing whereas ctDNA isolated from plasma sampled across the pre-treatment, response and eventual progression journey were analysed using the Guardant INFINITY platform equipped to detect mutations in &amp;gt;800 genes, and genome wide methylation, including promoter methylation in 398 cancer-related genes. Somatic mutations or regions of methylation that were associated with resistance were identified, including BRCA1/2 reversion mutations. Somatic mutations with the potential to cause PARPi resistance were annotated using CRISPR screen data and other functional analyses describing genes that alter PARPi synthetic lethality. Results: The most common resistance mechanism was BRCA1/2 reversion mutation (51%; n=8 BRCA1m patients, n=10 BRCA2m patients). Most reversions (77%) occurred via deletions (BRCA1, 70%; BRCA2, 79%) and exhibited microhomology use at junctions (&amp;gt; 80% for both BRCA1 and BRCA2). In 14 patients, multiple concurrent reversion mutations were detected with VAFs ranging from 0.1-40% for BRCA1 and 0.05-18% for BRCA2. Changes in VAF over time indicated that different reversions in the same patient may impart different fitness advantages in the face of treatment. Loss-of-function mutations in the 53BP1-Shieldin pathway were identified in two BRCA1m patients, and VUS mutations in the pathway were seen in two further patients. In some patients, reversion mutations co-occurred with either 53BP1-Shieldin pathway mutations or with replication fork stability mutations (PAXIP1 in BRCA2m patients) - indicating concurrent but mechanistically distinct forms of resistance develop in the same patient. We isolated a PDX model from one patient that exhibits resistance via both BRCA1 reversion and loss of 53BP1, allowing this phenomenon to be modelled in more detail. We also detected BRCA1 methylation in ctDNA in a patient with a somatic rearrangement in BRCA1. Conclusions: Liquid biopsy profiling of PARPi-resistance breast cancer patients indicates that many patients develop multiple reversion mutations, and that alterations in the 53BP1-Shieldin pathway are present but less frequent. The co-occurrence of 53BP1-Shieldin pathway mutations and reversion mutations suggests parallel mechanisms of resistance can operate in the same patient. Citation Format: Elizabeth Harvey-Jones, Maya Raghunandan, Luisa Robbez-Masson, Alaguthurai Thanussuyah, Roberta Liccardo, Arielle Yablonovitch, Mingyang Cai, Leylah Drusbosky, Michael Dorschner, Lorena Magraner Pardo, Rebecca Marlow, Asha Konde, Jennifer Trendell, John Alexander, Syed Haider, Chris Starling, Ioannis Roxanis, Jennifer Yen, Stephen J. Pettitt, Christopher J. Lord, Andrew N. Tutt. Longitudinal analysis of PARP inhibitor and platinum resistance in BRCA1/2m breast cancer using liquid biopsy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6094.

Cloning by SCNT: Integrating Technical and Biology-Driven Advances.

Somatic cell nuclear transfer (SCNT) into enucleated oocytes initiates nuclear reprogramming of lineage-committed cells to totipotency. Pioneer SCNT work culminated with cloned amphibians from tadpoles, while technical and biology-driven advances led to cloned mammals from adult animals. Cloning technology has been addressing fundamental questions in biology, propagating desired genomes, and contributing to the generation of transgenic animals or patient-specific stem cells. Nonetheless, SCNT remains technically complex and cloning efficiency relatively low. Genome-wide technologies revealed barriers to nuclear reprogramming, such as persistent epigenetic marks of somatic origin and reprogramming resistant regions of the genome. To decipher the rare reprogramming events that are compatible with full-term cloned development, it will likely require technical advances for large-scale production of SCNT embryos alongside extensive profiling by single-cell multi-omics. Altogether, cloning by SCNT remains a versatile technology, while further advances should continuously refresh the excitement of its applications.

Pilot study assessing the effect of Fascial Manipulation on fascial densifications and associated pain.

We assessed effectiveness of Fascial Manipulation (FM) in reducing densification thickness and associated acute pain in normal humans. Fascial densifications were identified using palpation and measured with diagnostic ultrasound within self-reported painful somatic regions. Pain intensity ratings were obtained in response to deep palpation of the self-reported painful somatic region before and after a brief FM intervention. Brief FM resulted in reduced densification thickness as well pain intensity. Sex differences were found neither in densification thickness nor pain intensity at any time point. However, a statistically significant positive correlation between densification thickness and pain intensity was observed in females but not males at both pre-FM and post-FM time points. As such, FM may be an effective therapeutic approach for acute pain associated with fascial densifications. While males and females exhibited comparable densification thickness and pain intensity levels at both pre-FM and post-FM time points, only females showed a statistically significant relationship between pain and densification, suggesting that females may be better able to perceive subtle differences in the magnitude of noxious sensory input.

History-dependent dopamine release increases cAMP levels in most basal amygdala glutamatergic neurons to control learning.

Dopaminergic inputs to basal amygdala (BA) instruct learning of motivational salience. This learning depends on intracellular plasticity signals such as cyclic adenosine monophosphate (cAMP), which is regulated by activation of dopamine receptors. We examine the dynamics of dopamine release and downstream signaling during multiple salient events occurring within tens of seconds. We perform real-time tracking and manipulation of cAMP in BA neurons invitro and invivo. Optogenetically evoked release of dopamine drives proportional increases in cAMP in almost all BA glutamatergic neurons, suggesting widespread actions of dopamine across neurons preferring positive or negative valence. This cAMP response decreases across trials with short intertrial intervals owing to depression of dopamine release. No such depression is evident when photostimulating cAMP production directly. cAMP and protein kinase A responses to repeated appetitive or aversive stimuli also exhibit pronounced depression. Thus, history-dependent dynamics of dopamine and cAMP may regulate learning of temporally clustered, salient stimuli.

The translatome of neuronal cell bodies, dendrites, and axons

To form synaptic connections and store information, neurons continuously remodel their proteomes. The impressive length of dendrites and axons imposes logistical challenges to maintain synaptic proteins at locations remote from the transcription source (the nucleus). The discovery of thousands of messenger RNAs (mRNAs) near synapses suggested that neurons overcome distance and gain autonomy by producing proteins locally. It is not generally known, however, if, how, and when localized mRNAs are translated into protein. To investigate the translational landscape in neuronal subregions, we performed simultaneous RNA sequencing (RNA-seq) and ribosome sequencing (Ribo-seq) from microdissected rodent brain slices to identify and quantify the transcriptome and translatome in cell bodies (somata) as well as dendrites and axons (neuropil). Thousands of transcripts were differentially translated between somatic and synaptic regions, with many scaffold and signaling molecules displaying increased translation levels in the neuropil. Most translational changes between compartments could be accounted for by differences in RNA abundance. Pervasive translational regulation was observed in both somata and neuropil influenced by specific mRNA features (e.g., untranslated region [UTR] length, RNA-binding protein [RBP] motifs, and upstream open reading frames [uORFs]). For over 800 mRNAs, the dominant source of translation was the neuropil. We constructed a searchable and interactive database for exploring mRNA transcripts and their translation levels in the somata and neuropil [MPI Brain Research, The mRNA translation landscape in the synaptic neuropil. https://public.brain.mpg.de/dashapps/localseq/ Accessed 5 October 2021]. Overall, our findings emphasize the substantial contribution of local translation to maintaining synaptic protein levels and indicate that on-site translational control is an important mechanism to control synaptic strength.

Dissecting Childhood B-Other Acute Lymphoblastic Leukemia: Lower Frequency of Druggable Gene Fusions in European Population?

Novel biological subtypes within B-other (B-o) acute lymphoblastic leukemia (ALL) have been described recently, based on the presence of genomic rearrangements (r.) of specific genes (DUX4 , ZNF384 and MEF2D ) or gene expression signatures (Ph-like and ETV6-RUNX1 -like ALL). Genomic profiling not only helped to dissect heterogeneity of B-o phenotype and outcome, but it also identified novel druggable aberrations such as kinase and cytokine receptor gene fusions, that were shown to occur mainly in Ph-like ALL. Unfortunately, only very limited population-based data are available to estimate an unbiased frequency of these novel genetic aberrations and subtypes, especially in European countries.We have analyzed a consecutive cohort of pediatric B-o ALL (negative for ETV6-RUNX1 , TCF3-PBX1 , BCR-ABL1 and KMT2A -involving fusions, hyper- and hypo-diploidy) diagnosed in the Czech Republic between 11/2012 and 5/2017. Transcriptome sequencing (RNAseq) was performed in 69/75 cases. High-density SNP array and whole exome sequencing were performed in 65 and 48 of those, respectively. Fusion transcripts, SNVs and indels were called from sequencing data using in-house pipeline based on publicly available software tools. Targeted analysis was performed to identify DUX4- r. Hierarchical clustering based on published gene sets defining DUX4 -r., Ph-like and ETV6-RUNX1 -like ALL was performed using RNAseq data. Somatic copy number aberrations and regions of uniparental disomy were called from SNP array data.Seventy five B-o cases represented 24% of all pediatric ALL (1-18 years) diagnosed within this period. According to hierarchical clustering, 23/69 cases were classified as DUX4 -r. ALL; all harbored DUX4 -r. They accounted for 33% of B-o ALL, representing a larger proportion than reported by others (Liu et al., EBioMedicine 2016; Lilljebjörn et al., Nat Commun 2016). Three cases (4%) and one case (1%) were positive for ZNF384-r. and MEF2D-r., respectively. Eleven cases (16%) were classified as Ph-like ALL; this subgroup included 7/9 cases with CRLF2 -r. and a single case with ETV6-ABL1 fusion. No other kinase/cytokine receptor gene fusion was detected. Four patients (6%) were classified as ETV6-RUNX1 -like ALL; all harbored aberrations of ETV6 . Strikingly, all 4 ETV6-RUNX1 -like cases displayed concurrently also Ph-like signature. The remaining 27 cases (39%) were not assigned to any novel subtype. Other lesions activating kinase signaling [mutations of NRAS (n=14), KRAS (n=7), FLT3 (n=4), PTPN11 (n=3), JAK2 (n=3), JAK1 (n=1), NF1 (n=1) and BRAF (n=1)] were found in 36 cases and were not enriched in Ph-like or other subtype. Of note, 9/27 of “unassigned” (but 0/42 of “assigned”) B-o harbored in-frame “non-kinase” PAX5 gene-involving fusions, both already described and novel, with largely unknown biological and clinical significance.Our data show that frequency of ZNF384 -r., MEF2D -r. and, importantly, also of kinase/cytokine receptor gene fusions (apart from CRLF2 -r.) might be lower in European population than estimated from data based on non-consecutive non-European cohorts (Liu et al.; Gu et al., Nat Commun 2016). To that extent our findings correspond with the recently published Swedish study (Lilljebjörn et al.); however, higher frequency of DUX4-r. ALL in our cohort indicates that differences in genetic composition of pediatric ALL may still occur even within Europe. Our findings highlight one of the flaws of Ph-like testing - non-specificity of Ph-like signature. Of note, the overlap of Ph-like and ETV6-RUNX1 -like signatures is also evidenced by positivity of Ph-like signature in ETV6-RUNX1 + ALL in study by Reshmi et al. (Blood 2017). Very low frequency of “kinase fusions” (apart from CRLF2 -r. which can be indirectly, yet reliably, diagnosed by flow cytometry) questions the use of Ph-like testing as a tool to pre-select ALL with high probability of having these lesions. Despite the use of complex genomics techniques a significant proportion of B-o ALL remains unclassified; interestingly, PAX5 fusions seem to be present in a significant proportion of them.To conclude, our population-based data confirm and specify novel genetic subtypes of pediatric B-o ALL; however, the low incidence of druggable kinase-activating fusions questions the extent of applicability of recently promoted individually “tailored” treatment.Support: AZV 15-30626A; AZV 16-30186A; GACR 15-06049Y; Primus/MED/28 DisclosuresNo relevant conflicts of interest to declare.

Limited Therapeutical Impact of Extensive Genomic Profiling in Relapsed Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia: Population-Based Four-Year Consecutive Cohort

High throughput genomic technologies, such as high-density SNP arrays and whole exome and transcriptome sequencing (WES, RNAseq), significantly deepened our insight into the genomic background and biology of acute lymphoblastic leukemia (ALL) during the past decade. Novel genetic aberrations and genomic and gene expression patterns have been described which led to the definition of several novel ALL subtypes and risk-predictive factors and to the identification of novel druggable and/or resistance-associated lesions potentially impacting therapy tailoring. Anecdotal evidence of therapeutical interventions based on the up-to-date genetic data has resulted into a great demand for these investigations. However, it is unclear what proportion of pediatric ALL patients, especially the cases of therapy-resistant leukemia, would really benefit from extensive genomic profiling.Between V/2013 and VIII/2017, relapse of Ph-negative B-cell precursor (BCP) ALL was diagnosed in 20 children (1-18 years) within the Czech Republic. In addition to the routine genetics, 18/20 cases were profiled by high density SNP array (18/18), WES (18/18) and RNAseq (17/18). The examined cohort comprised 10 B-other, 4 hyperdiploid, 3 ETV6-RUNX1 -positive and 1 KMT2A -rearranged ALL. At primary leukemia manifestation, all children were treated according to the ALL-BFM protocols [BFM 95 (n=1), BFM 2000 (n=1) and BFM 2009 (n=16)] and suffered very early (n=1), early (n=4) or late relapses (n=13). Upon disease relapse, 5, 11 and 2 cases were treated according to ALL-REZ BFM 2002 and IntReALL 2010 SR and HR protocols, respectively.Bulk bone marrow (BM) samples or sorted leukemic blasts were analyzed in isolated BM (n=15) and combined BM+CNS (n=2) relapses, while tissue from uterus infiltrated with blasts was analyzed in a single case of isolated extramedullary relapse. In addition to leukemic blasts, remission samples were analyzed by WES in all and by SNP array in selected cases to distinguish germline genetic variants. Somatic copy number aberrations and regions of uniparental disomy were called from SNP array data using commercial softwares. WES and RNAseq data were processed and somatic single nucleotide variants, indels and fusion transcripts were called using in-house pipeline based on publicly available software tools. Targeted analysis was performed to identify DUX4 gene rearrangements (r.). Hierarchical clustering based on gene expression profiles (unsupervised clustering and clustering with gene sets defining DUX4 -r., Ph-like and ETV6-RUNX1 -like ALL) was performed using RNAseq data. To facilitate this analysis, gene expression data from relapse cohort were combined with primary diagnosis data from >50 B-other ALL cases. Clinically important findings were reported to national study centre.Two B-other cases were classified as DUX4 -r. and 2 other cases as Ph-like ALL. Of note, 2 cases (both treated as standard risk in primary diagnosis) had iAMP21. Apart from 3 CRLF2 -rearrangements (1 present in Ph-like case), 6 KRAS and 2 NRAS genes mutations, no potentially druggable lesions were found. While the frequency of CRLF2 -rearrangements and RAS mutations found in relapse was comparable to that seen in primary disease, relapse cohort was enriched for lesions associated with unfavorable prognosis and therapy resistance. Three and four cases harbored deletion of TP53 gene and IKZF1 plus deletion pattern; however, in 1/2 cases with available data from primary diagnosis the IKZF1 plus pattern was identified only in relapse. Deletion of CREBBP and TBL1XR1 genes, potentially associated with resistance to glucocorticoids, were found in 2 cases and 1 case, respectively. Mutations of NT5C2 gene resulting in resistance to mercaptopurine were found in 2 cases. Three out of 18 analyzed cases did not harbor any of the above mentioned lesions. None of the genetic findings directly impacted patients' treatment.To conclude, we successfully implemented advanced genomics into centralized diagnostics of resistant ALL at the national level. Although it improved our understanding of disease relapse (yet, not in 100% of cases) the results were not translated to therapy tailoring in patients treated according to the large international protocols, thus, questioning the cost/benefit ratio of such extensive genetic diagnostics.Support: AZV 15-30626A and 16-30186A, GACR 15-06049Y, Primus/MED/28 DisclosuresNo relevant conflicts of interest to declare.

Morphological Heterogeneity of the Endoplasmic Reticulum within Neurons and Its Implications in Neurodegeneration.

The endoplasmic reticulum (ER) is a multipurpose organelle comprising dynamic structural subdomains, such as ER sheets and tubules, serving to maintain protein, calcium, and lipid homeostasis. In neurons, the single ER is compartmentalized with a careful segregation of the structural subdomains in somatic and neurite (axodendritic) regions. The distribution and arrangement of these ER subdomains varies between different neuronal types. Mutations in ER membrane shaping proteins and morphological changes in the ER are associated with various neurodegenerative diseases implying significance of ER morphology in maintaining neuronal integrity. Specific neurons, such as the highly arborized dopaminergic neurons, are prone to stress and neurodegeneration. Differences in morphology and functionality of ER between the neurons may account for their varied sensitivity to stress and neurodegenerative changes. In this review, we explore the neuronal ER and discuss its distinct morphological attributes and specific functions. We hypothesize that morphological heterogeneity of the ER in neurons is an important factor that accounts for their selective susceptibility to neurodegeneration.

ANMAF: an automated neuronal morphology analysis framework using convolutional neural networks

Measurement of neuronal size is challenging due to their complex histology. Current practice includes manual or pseudo-manual measurement of somatic areas, which is labor-intensive and prone to human biases and intra-/inter-observer variances. We developed a novel high-throughput neuronal morphology analysis framework (ANMAF), using convolutional neural networks (CNN) to automatically contour the somatic area of fluorescent neurons in acute brain slices. Our results demonstrate considerable agreements between human annotators and ANMAF on detection, segmentation, and the area of somatic regions in neurons expressing a genetically encoded fluorophore. However, in contrast to humans, who exhibited significant variability in repeated measurements, ANMAF produced consistent neuronal contours. ANMAF was generalizable across different imaging protocols and trainable even with a small number of humanly labeled neurons. Our framework can facilitate more rigorous and quantitative studies of neuronal morphology by enabling the segmentation of many fluorescent neurons in thick brain slices in a standardized manner.

Supraspinal Mechanisms of Intestinal Hypersensitivity.

Gut inflammation or injury causes intestinal hypersensitivity (IHS) and hyperalgesia, which can persist after the initiating pathology resolves, are often referred to somatic regions and exacerbated by psychological stress, anxiety or depression, suggesting the involvement of both the spinal cord and the brain. The supraspinal mechanisms of IHS remain to be fully elucidated, however, over the last decades the series of intestinal pathology-associated neuroplastic changes in the brain has been revealed, being potentially responsible for the phenomenon. This paper reviews current clinical and experimental data, including the authors' own findings, on these functional, structural, and neurochemical/molecular changes within cortical, subcortical and brainstem regions processing and modulating sensory signals from the gut. As concluded in the review, IHS can develop and maintain due to the bowel inflammation/injury-induced persistent hyperexcitability of viscerosensory brainstem and thalamic nuclei and sensitization of hypothalamic, amygdala, hippocampal, anterior insular, and anterior cingulate cortical areas implicated in the neuroendocrine, emotional and cognitive modulation of visceral sensation and pain. An additional contribution may come from the pathology-triggered dysfunction of the brainstem structures inhibiting nociception. The mechanism underlying IHS-associated regional hyperexcitability is enhanced NMDA-, AMPA- and group I metabotropic receptor-mediated glutamatergic neurotransmission in association with altered neuropeptide Y, corticotropin-releasing factor, and cannabinoid 1 receptor signaling. These alterations are at least partially mediated by brain microglia and local production of cytokines, especially tumor necrosis factor α. Studying the IHS-related brain neuroplasticity in greater depth may enable the development of new therapeutic approaches against chronic abdominal pain in inflammatory bowel disease.

Calcium imaging in population of dorsal root ganglion neurons unravels novel mechanisms of visceral pain sensitization and referred somatic hypersensitivity.

Mechanisms of visceral pain sensitization and referred somatic hypersensitivity remain unclear. We conducted calcium imaging in Pirt-GCaMP6s mice to gauge responses of dorsal root ganglion (DRG) neurons to visceral and somatic stimulation in vivo. Intracolonic instillation of 2,4,6-trinitrobenzene sulfonic acid (TNBS) induced colonic inflammation and increased the percentage of L6 DRG neurons that responded to colorectal distension above that of controls at day 7. Colorectal distension did not activate L4 DRG neurons. TNBS-treated mice exhibited more Evans blue extravasation than did control mice and developed mechanical hypersensitivity in low-back skin and hind paws, which are innervated by L6 and L4 DRG neurons, respectively, suggesting that colonic inflammation induced mechanical hypersensitivity in both homosegmental and heterosegmental somatic regions. Importantly, the percentage of L4 DRG neurons activated by hind paw pinch and brush stimulation and calcium responses of L6 DRG neurons to low-back brush stimulation were higher at day 7 after TNBS than those in control mice. Visceral irritation from intracolonic capsaicin instillation also increased Evans blue extravasation in hind paws and low-back skin and acutely increased the percentage of L4 DRG neurons responding to hind paw pinch and the response of L6 DRG neurons to low-back brush stimulation. These findings suggest that TNBS-induced colitis and capsaicin-induced visceral irritation may sensitize L6 DRG neurons to colorectal and somatic inputs and also increase the excitability of L4 DRG neurons that do not receive colorectal inputs. These changes may represent a potential peripheral neuronal mechanism for visceral pain sensitization and referred somatic hypersensitivity.

Regulation of Kappa Opioid Receptor Inactivation Depends on Sex and Cellular Site of Antagonist Action.

The prototypical member of the receptor-inactivating kappa opioid receptor (KOR) antagonists, norbinaltorphimine (norBNI), produces prolonged receptor inactivation by a cJun kinase mechanism. These antagonists have potential therapeutic utility in the treatment of stress disorders; however, additional preclinical characterization is necessary to understand important aspects of their action. In this study, we report that norBNI does not work as effectively in female mice as in males because of estrogen regulation of G protein receptor kinase (GRK); pretreatment of ovary-intact female mice with the selective GRK2/3 inhibitor, Compound 101, made females equally sensitive to norBNI as males. Prior observations suggested that in vivo treatment with norBNI does not produce long-lasting inhibition of KOR regulation of dopamine release in the nucleus accumbens. We assessed the persistence of norBNI receptor inactivation in subcellular compartments. Fast-scan cyclic voltammetry recordings confirmed that presynaptic inhibition of dopamine release by the KOR agonist U69,593 was not blocked by in vivo pretreatment with norBNI under conditions that prevented KOR-mediated aversion and analgesia. We employed a novel in vivo proxy sensor of KOR activation, adenovirus associated double floxed inverted-HyPerRed, and demonstrated that KOR activation stimulates cJun kinase-dependent reactive oxygen species (ROS) production in somatic regions of ventral tegmental area dopamine neurons, but did not activate ROS production in dopamine terminals. The compartment selective action helps explain how dopamine somatic, but not terminally expressed, KORs are inactivated by norBNI. These results further elucidate molecular signaling mechanisms mediating receptor-inactivating KOR antagonist action and advance medication development for this novel class of stress-resilience medications. SIGNIFICANCE STATEMENT: Kappa opioid receptor (KOR) antagonists are being developed as novel proresilience therapeutics for the treatment of mood and substance use disorders. This study showed that the long-acting KOR antagonists are affected by both the sex of the animal and the subcellular compartment in which the receptor is expressed.

Body Surface Examination Facilitated by Digital Microscopy

Patients present daily with conditions affecting the superficial surface of the body of all somatic regions. From scalp hair, skin, and ocular structures all the way to the anal sphincter. In this regard, elusive clinical detail that is imperceptible to the naked eye may be revealed through

Nervous system-wide profiling of presynaptic mRNAs reveals regulators of associative memory

Presynaptic protein synthesis is important in the adult central nervous system; however, the nervous system-wide set of mRNAs localized to presynaptic areas has yet to be identified in any organism. Here we differentially labeled somatic and synaptic compartments in adult C. elegans with fluorescent proteins, and isolated synaptic and somatic regions from the same population of animals. We used this technique to determine the nervous system-wide presynaptic transcriptome by deep sequencing. Analysis of the synaptic transcriptome reveals that synaptic transcripts are predicted to have specialized functions in neurons. Differential expression analysis identified 542 genes enriched in synaptic regions relative to somatic regions, with synaptic functions conserved in higher organisms. We find that mRNAs for pumilio RNA-binding proteins are abundant in synaptic regions, which we confirmed through high-sensitivity in situ hybridization. Presynaptic PUMILIOs regulate associative memory. Our approach enables the identification of new mechanisms that regulate synaptic function and behavior.

The Altered Somatic Brain Network in State Anxiety.

Highly anxious individuals often show excessive emotional arousal, somatic arousal, and characteristics of mental illness. Previous researches have extensively investigated the emotional and cognitive biases of individuals with high anxiety, but overlooked the spontaneous brain activity and functional connections associated with somatic arousal. In this study, we investigated the relationship between state anxiety and the spontaneous brain activity of the somatosensory cortex in a non-clinical healthy population with state anxiety. Furthermore, we also explored the functional connections of the somatosensory cortex. We found that state anxiety was positively correlated with the amplitude of low-frequency fluctuations (ALFFs) of somatic related brain regions, including the right postcentral gyrus (somatosensory cortex) and the right precentral gyrus (somatic motor cortex). Furthermore, we found that state anxiety was positively correlated with the connections between the postcentral gyrus and the left cerebellum gyrus, whereas state anxiety was negatively correlated with the connectivity between the postcentral gyrus and brain regions including the left inferior frontal cortex and left medial superior frontal cortex. These results revealed the association between the anxious individuals’ body-loop and state anxiety in a healthy population, which revealed the importance of somatic brain regions in anxiety symptoms and provided a new perspective on anxiety for further study.