Profiling learning strategies of medical students: A person-centered approach.

BACKGROUND: Students within a cohort might employ unique subsets of learning strategies (LS) to study. However, little research has aimed to elucidate subgroup-specific LS usage among medical students. Recent methodological developments, particularly person-centred approaches such as latent profile analysis (LPA), offer ways to identify relevant subgroups with dissimilar patterns of LS use. In this paper, we apply LPA to explore subgroups of medical students during preclinical training in anatomy and examine how these patterns are linked with learning outcomes. METHODS: We analysed the LS used by 689 undergraduate, 1st and 2nd-year medical students across 6 German universities who completed the short version of the Learning Strategies of University Students (LIST-K) questionnaire, and answered questions towards external criteria such as learning resources and performance. We used the thirteen different LS facets of the LIST-K (four cognitive, three metacognitive, three management of internal and three management of external resources) as LPA indicators. RESULTS: Based on LPA, students can be grouped into four distinct learning profiles: Active learners (45% of the cohort), collaborative learners (17%), structured learners (29%) and passive learners (9%). Students in each of those latent profiles combine the 13 LS facets in a unique way to study anatomy. The profiles differ in both, the overall level of LS usage, and unique combinations of LS used for learning. Importantly, we find that the facets of LS show heterogeneous and subgroup-specific correlations with relevant outcome criteria, which partly overlap but mostly diverge from effects observed on the population level. CONCLUSIONS: The effects observed by LPA expand results from variable-centered efforts and challenge the notion that LS operate on a linear continuum. These results highlight the heterogeneity between subgroups of learners and help generate a more nuanced interpretation of learning behaviour. Lastly, our analysis offers practical implications for educators seeking to tailor learning experiences to meet individual student needs.

Regulation of hippocampal mossy fiber-CA3 synapse function by a Bcl11b/C1ql2/Nrxn3(25b+) pathway.

The transcription factor Bcl11b has been linked to neurodevelopmental and neuropsychiatric disorders associated with synaptic dysfunction. Bcl11b is highly expressed in dentate gyrus granule neurons and is required for the structural and functional integrity of mossy fiber-CA3 synapses. The underlying molecular mechanisms, however, remained unclear. We show in mice that the synaptic organizer molecule C1ql2 is a direct functional target of Bcl11b that regulates synaptic vesicle recruitment and long-term potentiation at mossy fiber-CA3 synapses in vivo and in vitro. Furthermore, we demonstrate C1ql2 to exert its functions through direct interaction with a specific splice variant of neurexin-3, Nrxn3(25b+). Interruption of C1ql2-Nrxn3(25b+) interaction by expression of a non-binding C1ql2 mutant or by deletion of Nrxn3 in the dentate gyrus granule neurons recapitulates major parts of the Bcl11b as well as C1ql2 mutant phenotype. Together, this study identifies a novel C1ql2-Nrxn3(25b+)-dependent signaling pathway through which Bcl11b controls mossy fiber-CA3 synapse function. Thus, our findings contribute to the mechanistic understanding of neurodevelopmental disorders accompanied by synaptic dysfunction.

The human brain contains billions of neurons working together to process the vast array of information we receive from our environment. These neurons communicate at junctions known as synapses, where chemical packages called vesicles released from one neuron stimulate a response in another. This synaptic communication is crucial for our ability to think, learn and remember. However, this activity depends on a complex interplay of proteins, whose balance and location within the neuron are tightly controlled. Any disruption to this delicate equilibrium can cause significant problems, including neurodevelopmental and neuropsychiatric disorders, such as schizophrenia and intellectual disability. One key regulator of activity at the synapse is a protein called Bcl11b, which has been linked to conditions affected by synaptic dysfunction. It plays a critical role in maintaining specific junctions known as mossy fibre synapses, which are important for learning and memory. One of the genes regulated by Bcl11b is C1ql2, which encodes for a synaptic protein. However, it is unclear what molecular mechanisms Bcl11b uses to carry out this role. To address this, Koumoundourou et al. explored the role of C1ql2 in mossy fibre synapses of adult mice. Experiments to manipulate the production of C1ql2 independently of Bcl11b revealed that C1ql2 is vital for recruiting vesicles to the synapse and strengthening synaptic connections between neurons. Further investigation showed that C1ql2's role in this process relies on interacting with another synaptic protein called neurexin-3. Disrupting this interaction reduced the amount of C1ql2 at the synapse and, consequently, impaired vesicle recruitment. These findings will help our understanding of how neurodevelopmental and neuropsychiatric disorders develop. Bcl11b, C1ql2 and neurexin-3 have been independently associated with these conditions, and the now-revealed interactions between these proteins offer new insights into the molecular basis of synaptic faults. This research opens the door to further study of how these proteins interact and their roles in brain health and disease.

Distinct Alterations in Dendritic Spine Morphology in the Absence of ß-Neurexins.

Dendritic spines are essential for synaptic function because they constitute the postsynaptic compartment of the neurons that receives the most excitatory input. The extracellularly shorter variant of the presynaptic cell adhesion molecules neurexins, ß-neurexin, has been implicated in various aspects of synaptic function, including neurotransmitter release. However, its role in developing or stabilizing dendritic spines as fundamental computational units of excitatory synapses has remained unclear. Here, we show through morphological analysis that the deletion of ß-neurexins in hippocampal neurons in vitro and in hippocampal tissue in vivo affects presynaptic dense-core vesicles, as hypothesized earlier, and, unexpectedly, alters the postsynaptic spine structure. Specifically, we observed that the absence of ß-neurexins led to an increase in filopodial-like protrusions in vitro and more mature mushroom-type spines in the CA1 region of adult knockout mice. In addition, the deletion of ß-neurexins caused alterations in the spine head dimension and an increase in spines with perforations of their postsynaptic density but no changes in the overall number of spines or synapses. Our results indicate that presynaptic ß-neurexins play a role across the synaptic cleft, possibly by aligning with postsynaptic binding partners and glutamate receptors via transsynaptic columns.

Transfer of learning in histology: Insights from a longitudinal study.

All anatomical educators hope that students apply past training to both similar and new tasks. This two-group longitudinal study investigated the development of such transfer of learning in a histology course. After 0, 10, and 20 sessions of the 10-week-long course, medical students completed theoretical tasks, examined histological slides trained in the course (retention task), and unfamiliar histological slides (transfer task). The results showed that students in the histology group gradually outperformed the control group in all tasks, especially in the second half of the course, η2 = 0.268 (p < 0.001). The best predictor of final transfer performance was students' retention performance after 10 sessions, ß = 0.32 (p = 0.028), and theoretical knowledge after 20 sessions, ß = 0.46 (p = 0.003). Results of eye tracking methodology further revealed that the histology group engaged in greater "visual activity" when solving transfer tasks, as indicated by an increase in the total fixation count, η2 = 0.103 (p = 0.014). This longitudinal study provides evidence that medical students can use what they learn in histology courses to solve unfamiliar problems but cautions that positive transfer effects develop relatively late in the course. Thus, course time and the complex relationship between theory, retention, and transfer holds critical implications for anatomical curricula seeking to foster the transfer of learning.

Webcam-based eye-tracking to measure visual expertise of medical students during online histology training.

Objectives: Visual expertise is essential for image-based tasks that rely on visual cues, such as in radiology or histology. Studies suggest that eye movements are related to visual expertise and can be measured by near-infrared eye-tracking. With the popularity of device-embedded webcam eye-tracking technology, cost-effective use in educational contexts has recently become amenable. This study investigated the feasibility of such methodology in a curricular online-only histology course during the 2021 summer term. Methods: At two timepoints (t1 and t2), third-semester medical students were asked to diagnose a series of histological slides while their eye movements were recorded. Students' eye metrics, performance and behavioral measures were analyzed using variance analyses and multiple regression models. Results: First, webcam-eye tracking provided eye movement data with satisfactory quality (mean accuracy=115.7 px±31.1). Second, the eye movement metrics reflected the students' proficiency in finding relevant image sections (fixation count on relevant areas=6.96±1.56 vs. irrelevant areas=4.50±1.25). Third, students' eye movement metrics successfully predicted their performance (R2adj=0.39, p<0.001). Conclusion: This study supports the use of webcam-eye-tracking expanding the range of educational tools available in the (digital) classroom. As the students' interest in using the webcam eye-tracking was high, possible areas of implementation will be discussed.

Medical imaging training with eye movement modeling examples: A randomized controlled study.

PURPOSE: To determine whether ultrasound training in which the expert's eye movements are superimposed to the underlying ultrasound video (eye movement modeling examples; EMMEs) leads to better learner outcomes than traditional eye movement-free instructions. MATERIALS AND METHODS: 106 undergraduate medical students were randomized in two groups; 51 students in the EMME group watched 5-min ultrasound examination videos combined with the eye movements of an expert performing the task. The identical videos without the eye movements were shown to 55 students in the control group. Performance and behavioral parameters were compared prepost interventional using ANOVAs. Additionally, cognitive load, and prior knowledge in anatomy were surveyed. RESULTS: After training, the EMME group identified more sonoanatomical structures correctly, and completed the tasks faster than the control group. This effect was partly mediated by a reduction of extraneous cognitive load. Participants with greater prior anatomical knowledge benefited the most from the EMME training. CONCLUSION: Displaying experts' eye movements in medical imaging training appears to be an effective way to foster medical interpretation skills of undergraduate medical students. One underlying mechanism might be that practicing with eye movements reduces cognitive load and helps learners activate their prior knowledge.

Concerted roles of LRRTM1 and SynCAM 1 in organizing prefrontal cortex synapses and cognitive functions.

Multiple trans-synaptic complexes organize synapse development, yet their roles in the mature brain and cooperation remain unclear. We analyzed the postsynaptic adhesion protein LRRTM1 in the prefrontal cortex (PFC), a region relevant to cognition and disorders. LRRTM1 knockout (KO) mice had fewer synapses, and we asked whether other synapse organizers counteract further loss. This determined that the immunoglobulin family member SynCAM 1 controls synapse number in PFC and was upregulated upon LRRTM1 loss. Combined LRRTM1 and SynCAM 1 deletion substantially lowered dendritic spine number in PFC, but not hippocampus, more than the sum of single KO impairments. Their cooperation extended presynaptically, and puncta of Neurexins, LRRTM1 partners, were less abundant in double KO (DKO) PFC. Electrophysiology and fMRI demonstrated aberrant neuronal activity in DKO mice. Further, DKO mice were impaired in social interactions and cognitive tasks. Our results reveal concerted roles of LRRTM1 and SynCAM 1 across synaptic, network, and behavioral domains.

Are stereotypes in decline? The portrayal of female anatomy in e-learning.

Sex and gender bias in anatomy learning materials are considered a "hidden obstacle" to gender equity in medical curricula. The purpose of this study was to investigate whether quantitative sex and gender biases do exist in popular anatomy e-learning platforms and compare the results with those found in contemporary textbooks and atlases. A systematic content-analysis was performed on N = 3767 images published from 2008 to 2021 in which sex/gender could be identified by considering technical aspects of illustration and various intersectional categories. E-learning platforms took into account an appropriate representation of the female body and presented even more females (n = 932/1412; 66%), more frequently from a ventral/anterior (χ2  = 26, P < 0.001) and whole-body perspective (χ2  = 27, P < 0.001). This was in contrast to German anatomy books, where the results pointed to a significant sex and gender bias. For example, all books assessed underrepresented females (n = 707/2355; 30%) and placed them in stereotypical sex-specific context (χ2  = 348, P < 0.001), showing them more often from a caudal/inferior (χ2  = 99, P < 0.001) and internal (χ2  = 132, P < 0.001) perspective. Altogether, the visual representation of sex and gender in anatomical curricula is still biased and the stereotypical perceptions of human anatomy seem to be a global issue. However, the increasing use of electronic learning platforms, which gradually replace traditional books is changing the way the male and female body is depicted, which might offer new opportunities for reducing stereotypes in anatomy education.

α2δ-4 and Cachd1 Proteins Are Regulators of Presynaptic Functions.

The α2δ auxiliary subunits of voltage-gated calcium channels (VGCC) were traditionally regarded as modulators of biophysical channel properties. In recent years, channel-independent functions of these subunits, such as involvement in synapse formation, have been identified. In the central nervous system, α2δ isoforms 1, 2, and 3 are strongly expressed, regulating glutamatergic synapse formation by a presynaptic mechanism. Although the α2δ-4 isoform is predominantly found in the retina with very little expression in the brain, it was recently linked to brain functions. In contrast, Cachd1, a novel α2δ-like protein, shows strong expression in brain, but its function in neurons is not yet known. Therefore, we aimed to investigate the presynaptic functions of α2δ-4 and Cachd1 by expressing individual proteins in cultured hippocampal neurons. Both α2δ-4 and Cachd1 are expressed in the presynaptic membrane and could rescue a severe synaptic defect present in triple knockout/knockdown neurons that lacked the α2δ-1-3 isoforms (α2δ TKO/KD). This observation suggests that presynaptic localization and the regulation of synapse formation in glutamatergic neurons is a general feature of α2δ proteins. In contrast to this redundant presynaptic function, α2δ-4 and Cachd1 differentially regulate the abundance of presynaptic calcium channels and the amplitude of presynaptic calcium transients. These functional differences may be caused by subtle isoform-specific differences in α1-α2δ protein-protein interactions, as revealed by structural homology modelling. Taken together, our study identifies both α2δ-4 and Cachd1 as presynaptic regulators of synapse formation, differentiation, and calcium channel functions that can at least partially compensate for the loss of α2δ-1-3. Moreover, we show that regulating glutamatergic synapse formation and differentiation is a critical and surprisingly redundant function of α2δ and Cachd1.

PI(4,5)P2 controls slit diaphragm formation and endocytosis in Drosophila nephrocytes.

Drosophila nephrocytes are an emerging model system for mammalian podocytes and proximal tubules as well as for the investigation of kidney diseases. Like podocytes, nephrocytes exhibit characteristics of epithelial cells, but the role of phospholipids in polarization of these cells is yet unclear. In epithelia, phosphatidylinositol(4,5)bisphosphate (PI(4,5)P2) and phosphatidylinositol(3,4,5)-trisphosphate (PI(3,4,5)P3) are asymmetrically distributed in the plasma membrane and determine apical-basal polarity. Here, we demonstrate that both phospholipids are present in the plasma membrane of nephrocytes, but only PI(4,5)P2 accumulates at slit diaphragms. Knockdown of Skittles, a phosphatidylinositol(4)phosphate 5-kinase, which produces PI(4,5)P2, abolished slit diaphragm formation and led to strongly reduced endocytosis. Notably, reduction in PI(3,4,5)P3 by overexpression of PTEN or expression of a dominant-negative phosphatidylinositol-3-kinase did not affect nephrocyte function, whereas enhanced formation of PI(3,4,5)P3 by constitutively active phosphatidylinositol-3-kinase resulted in strong slit diaphragm and endocytosis defects by ectopic activation of the Akt/mTOR pathway. Thus, PI(4,5)P2 but not PI(3,4,5)P3 is essential for slit diaphragm formation and nephrocyte function. However, PI(3,4,5)P3 has to be tightly controlled to ensure nephrocyte development.

"Fun slipping into the doctor's role"-The relationship between sonoanatomy teaching and professional identity formation before and during the Covid-19 pandemic.

The various psychological dimensions of professional identity formation (PIF) are an important aspect of the study course for undergraduate medical students. Anatomical learning environments have been repeatedly shown to play a critical role in forming such an identity; however, relevance of PIF during sonoanatomical training remains underexplored. At the end of their basic anatomy studies, third-semester medical students took part in a four-day block course on anatomy and imaging. Anatomical content was revised in small groups using peer teaching and imaging methods, including one hour of hands-on sonoanatomy sessions each day. On-site sonoanatomy was identified as an excellent format to support students' transition from the pre-clinical to clinical phase as medical experts-to-be. Students enjoyed practical exercises and the clinical input, which increased their interest in the medical profession and their academic studies. This study further examined the effects of the transition into an online-only format, necessitated by the current Covid-19 pandemic. A comparison was made between the quantitative and qualitative evaluation data, and the written results of examinations of several on-site (n = 1096, mean age = 22.4 years ± 2.18), and online-only cohorts (n = 230, mean age = 22.6 years ± 2.21). The online-only transition led to a reduction of all PIF-related variables measured, losing identity-related variables, increasing students' stress levels, and reducing their long-term academic performance. Together, this study demonstrates presence of PIF in undergraduate sonoanatomy teaching, and cautions against the uncritical online-only substitution of hands-on learning environments.

Neurexin 1 variants as risk factors for suicide death.

Suicide is a significant public health concern with complex etiology. Although the genetic component of suicide is well established, the scope of gene networks and biological mechanisms underlying suicide has yet to be defined. Previously, we reported genome-wide evidence that neurexin 1 (NRXN1), a key synapse organizing molecule, is associated with familial suicide risk. Here we present new evidence for two non-synonymous variants (rs78540316; P469S and rs199784139; H885Y) associated with increased familial risk of suicide death. We tested the impact of these variants on binding interactions with known partners and assessed functionality in a hemi-synapse formation assay. Although the formation of hemi-synapses was not altered with the P469S variant relative to wild-type, both variants increased binding to the postsynaptic binding partner, leucine-rich repeat transmembrane neuronal 2 (LRRTM2) in vitro. Our findings indicate that variants in NRXN1 and related synaptic genes warrant further study as risk factors for suicide death.

Endogenous ß-neurexins on axons and within synapses show regulated dynamic behavior.

Neurexins are key organizer molecules that regulate synaptic function and are implicated in autism and schizophrenia. ß-neurexins interact with numerous cell adhesion and receptor molecules, but their neuronal localization remains elusive. Using single-molecule tracking and high-resolution microscopy to detect neurexin1ß and neurexin3ß in primary hippocampal neurons from knockin mice, we demonstrate that endogenous ß-neurexins are present in fewer than half of excitatory and inhibitory synapses. Moreover, we observe a large extrasynaptic pool of ß-neurexins on axons and show that axonal ß-neurexins diffuse with higher surface mobility than those transiently confined within synapses. Stimulation of neuronal activity further increases the mobility of synaptic and axonal ß-neurexins, whereas inhibition causes the opposite. Blocking ectodomain cleavage by metalloproteases also reduces ß-neurexin mobility and enhances glutamate release. These findings suggest that the surface mobility of endogenous ß-neurexins inside and outside of synapses is dynamically regulated and linked to neuronal activity.

Presynaptic α2δ subunits are key organizers of glutamatergic synapses.

In nerve cells the genes encoding for α2δ subunits of voltage-gated calcium channels have been linked to synaptic functions and neurological disease. Here we show that α2δ subunits are essential for the formation and organization of glutamatergic synapses. Using a cellular α2δ subunit triple-knockout/knockdown model, we demonstrate a failure in presynaptic differentiation evidenced by defective presynaptic calcium channel clustering and calcium influx, smaller presynaptic active zones, and a strongly reduced accumulation of presynaptic vesicle-associated proteins (synapsin and vGLUT). The presynaptic defect is associated with the downscaling of postsynaptic AMPA receptors and the postsynaptic density. The role of α2δ isoforms as synaptic organizers is highly redundant, as each individual α2δ isoform can rescue presynaptic calcium channel trafficking and expression of synaptic proteins. Moreover, α2δ-2 and α2δ-3 with mutated metal ion-dependent adhesion sites can fully rescue presynaptic synapsin expression but only partially calcium channel trafficking, suggesting that the regulatory role of α2δ subunits is independent from its role as a calcium channel subunit. Our findings influence the current view on excitatory synapse formation. First, our study suggests that postsynaptic differentiation is secondary to presynaptic differentiation. Second, the dependence of presynaptic differentiation on α2δ implicates α2δ subunits as potential nucleation points for the organization of synapses. Finally, our results suggest that α2δ subunits act as transsynaptic organizers of glutamatergic synapses, thereby aligning the synaptic active zone with the postsynaptic density.

Apical-basal polarity regulators are essential for slit diaphragm assembly and endocytosis in Drosophila nephrocytes.

Apical-basal polarity is a key feature of most epithelial cells and it is regulated by highly conserved protein complexes. In mammalian podocytes, which emerge from columnar epithelial cells, this polarity is preserved and the tight junctions are converted to the slit diaphragms, establishing the filtration barrier. In Drosophila, nephrocytes show several structural and functional similarities with mammalian podocytes and proximal tubular cells. However, in contrast to podocytes, little is known about the role of apical-basal polarity regulators in these cells. In this study, we used expansion microscopy and found the apical polarity determinants of the PAR/aPKC and Crb-complexes to be predominantly targeted to the cell cortex in proximity to the nephrocyte diaphragm, whereas basolateral regulators also accumulate intracellularly. Knockdown of PAR-complex proteins results in severe endocytosis and nephrocyte diaphragm defects, which is due to impaired aPKC recruitment to the plasma membrane. Similar, downregulation of most basolateral polarity regulators disrupts Nephrin localization but had surprisingly divergent effects on endocytosis. Our findings suggest that morphology and slit diaphragm assembly/maintenance of nephrocytes is regulated by classical apical-basal polarity regulators, which have distinct functions in endocytosis.

Missense mutations in CASK, coding for the calcium-/calmodulin-dependent serine protein kinase, interfere with neurexin binding and neurexin-induced oligomerization.

Mutations in the X-linked gene coding for the calcium-/calmodulin-dependent serine protein kinase (CASK) are associated with severe neurological disorders ranging from intellectual disability (in males) to mental retardation and microcephaly with pontine and cerebellar hypoplasia. CASK is involved in transcription control, in the regulation of trafficking of the post-synaptic NMDA and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, and acts as a presynaptic scaffolding protein. For CASK missense mutations, it is mostly unclear which of CASK's molecular interactions and cellular functions are altered and contribute to patient phenotypes. We identified five CASK missense mutations in male patients affected by neurodevelopmental disorders. These and five previously reported mutations were systematically analysed with respect to interaction with CASK interaction partners by co-expression and co-immunoprecipitation. We show that one mutation in the L27 domain interferes with binding to synapse-associated protein of 97 kDa. Two mutations in the guanylate kinase (GK) domain affect binding of CASK to the nuclear factors CASK-interacting nucleosome assembly protein (CINAP) and T-box, brain, 1 (Tbr1). A total of five mutations in GK as well as PSD-95/discs large/ZO-1 (PDZ) domains affect binding of CASK to the pre-synaptic cell adhesion molecule Neurexin. Upon expression in neurons, we observe that binding to Neurexin is not required for pre-synaptic localization of CASK. We show by bimolecular fluorescence complementation assay that Neurexin induces oligomerization of CASK, and that mutations in GK and PDZ domains interfere with the Neurexin-induced oligomerization of CASK. Our data are supported by molecular modelling, where we observe that the cooperative activity of PDZ, SH3 and GK domains is required for Neurexin binding and oligomerization of CASK.

Deletion of ß-Neurexins in Mice Alters the Distribution of Dense-Core Vesicles in Presynapses of Hippocampal and Cerebellar Neurons.

Communication between neurons through synapses includes the release of neurotransmitter-containing synaptic vesicles (SVs) and of neuromodulator-containing dense-core vesicles (DCVs). Neurexins (Nrxns), a polymorphic family of cell surface molecules encoded by three genes in vertebrates (Nrxn1-3), have been proposed as essential presynaptic organizers and as candidates for cell type-specific or even synapse-specific regulation of synaptic vesicle exocytosis. However, it remains unknown whether Nrxns also regulate DCVs. Here, we report that at least ß-neurexins (ß-Nrxns), an extracellularly smaller Nrxn variant, are involved in the distribution of presynaptic DCVs. We found that conditional deletion of all three ß-Nrxn isoforms in mice by lentivirus-mediated Cre recombinase expression in primary hippocampal neurons reduces the number of ultrastructurally identified DCVs in presynaptic boutons. Consistently, colabeling against marker proteins revealed a diminished population of chromogranin A- (ChrgA-) positive DCVs in synapses and axons of ß-Nrxn-deficient neurons. Moreover, we validated the impaired DCV distribution in cerebellar brain tissue from constitutive ß-Nrxn knockout (ß-TKO) mice, where DCVs are normally abundant and ß-Nrxn isoforms are prominently expressed. Finally, we observed that the ultrastructure and marker proteins of the Golgi apparatus, responsible for packaging neuropeptides into DCVs, seem unchanged. In conclusion, based on the validation from the two deletion strategies in conditional and constitutive KO mice, two neuronal populations from the hippocampus and cerebellum, and two experimental protocols in cultured neurons and in the brain tissue, this study presented morphological evidence that the number of DCVs at synapses is altered in the absence of ß-Nrxns. Our results therefore point to an unexpected contribution of ß-Nrxns to the organization of neuropeptide and neuromodulator function, in addition to their more established role in synaptic vesicle release.

An extracellular vesicle-related gene expression signature identifies high-risk patients in medulloblastoma.

BACKGROUND: Medulloblastoma (MB) is a malignant brain tumor in childhood. It comprises 4 subgroups with different clinical behaviors. The aim of this study was to characterize the transcriptomic landscape of MB, both at the level of individual tumors as well as in large patient cohorts. METHODS: We used a combination of single-cell transcriptomics, cell culture models and biophysical methods such as nanoparticle tracking analysis and electron microscopy to investigate intercellular communication in the MB tumor niche. RESULTS: Tumor cells of the sonic hedgehog (SHH)-MB subgroup show a differentiation blockade. These cells undergo extensive metabolic reprogramming. The gene expression profiles of individual tumor cells show a partial convergence with those of tumor-associated glial and immune cells. One possible cause is the transfer of extracellular vesicles (EVs) between cells in the tumor niche. We were able to detect EVs in co-culture models of MB tumor cells and oligodendrocytes. We also identified a gene expression signature, EVS, which shows overlap with the proteome profile of large oncosomes from prostate cancer cells. This signature is also present in MB patient samples. A high EVS expression is one common characteristic of tumors that occur in high-risk patients from different MB subgroups or subtypes. CONCLUSIONS: With EVS, our study uncovered a novel gene expression signature that has a high prognostic significance across MB subgroups.

Enhanced LTP of population spikes in the dentate gyrus of mice haploinsufficient for neurobeachin.

Deletion of the autism candidate molecule neurobeachin (Nbea), a large PH-BEACH-domain containing neuronal protein, has been shown to affect synaptic function by interfering with neurotransmitter receptor targeting and dendritic spine formation. Previous analysis of mice lacking one allele of the Nbea gene identified impaired spatial learning and memory in addition to altered autism-related behaviours. However, no functional data from living heterozygous Nbea mice (Nbea+/-) are available to corroborate the behavioural phenotype. Here, we explored the consequences of Nbea haploinsufficiency on excitation/inhibition balance and synaptic plasticity in the intact hippocampal dentate gyrus of Nbea+/- animals in vivo by electrophysiological recordings. Based on field potential recordings, we show that Nbea+/- mice display enhanced LTP of the granule cell population spike, but no differences in basal synaptic transmission, synapse numbers, short-term plasticity, or network inhibition. These data indicate that Nbea haploinsufficiency causes remarkably specific alterations to granule cell excitability in vivo, which may contribute to the behavioural abnormalities in Nbea+/- mice and to related symptoms in patients.

Auxiliary α2δ1 and α2δ3 Subunits of Calcium Channels Drive Excitatory and Inhibitory Neuronal Network Development.

VGCCs are multisubunit complexes that play a crucial role in neuronal signaling. Auxiliary α2δ subunits of VGCCs modulate trafficking and biophysical properties of the pore-forming α1 subunit and trigger excitatory synaptogenesis. Alterations in the expression level of α2δ subunits were implicated in several syndromes and diseases, including chronic neuropathic pain, autism, and epilepsy. However, the contribution of distinct α2δ subunits to excitatory/inhibitory imbalance and aberrant network connectivity characteristic for these pathologic conditions remains unclear. Here, we show that α2δ1 overexpression enhances spontaneous neuronal network activity in developing and mature cultures of hippocampal neurons. In contrast, overexpression, but not downregulation, of α2δ3 enhances neuronal firing in immature cultures, whereas later in development it suppresses neuronal activity. We found that α2δ1 overexpression increases excitatory synaptic density and selectively enhances presynaptic glutamate release, which is impaired on α2δ1 knockdown. Overexpression of α2δ3 increases the excitatory synaptic density as well but also facilitates spontaneous GABA release and triggers an increase in the density of inhibitory synapses, which is accompanied by enhanced axonaloutgrowth in immature interneurons. Together, our findings demonstrate that α2δ1 and α2δ3 subunits play distinct but complementary roles in driving formation of structural and functional network connectivity during early development. An alteration in α2δ surface expression during critical developmental windows can therefore play a causal role and have a profound impact on the excitatory-to-inhibitory balance and network connectivity.SIGNIFICANCE STATEMENT The computational capacity of neuronal networks is determined by their connectivity. Chemical synapses are the main interface for transfer of information between individual neurons. The initial formation of network connectivity requires spontaneous electrical activity and the calcium channel-mediated signaling. We found that, in early development, auxiliary α2δ3 subunits of calcium channels foster presynaptic release of GABA, trigger formation of inhibitory synapses, and promote axonal outgrowth in inhibitory interneurons. In contrast, later in development, α2δ1 subunits promote the glutamatergic neurotransmission and synaptogenesis, as well as strongly enhance neuronal network activity. We propose that formation of connectivity in neuronal networks is associated with a concerted interplay of α2δ1 and α2δ3 subunits of calcium channels.