Combined expansion and STED microscopy reveals altered fingerprints of postsynaptic nanostructure across brain regions in ASD-related SHANK3-deficiency.

Synaptic dysfunction is a key feature of SHANK-associated disorders such as autism spectrum disorder, schizophrenia, and Phelan-McDermid syndrome. Since detailed knowledge of their effect on synaptic nanostructure remains limited, we aimed to investigate such alterations in ex11|SH3 SHANK3-KO mice combining expansion and STED microscopy. This enabled high-resolution imaging of mosaic-like arrangements formed by synaptic proteins in both human and murine brain tissue. We found distinct shape-profiles as fingerprints of the murine postsynaptic scaffold across brain regions and genotypes, as well as alterations in the spatial and molecular organization of subsynaptic domains under SHANK3-deficient conditions. These results provide insights into synaptic nanostructure in situ and advance our understanding of molecular mechanisms underlying synaptic dysfunction in neuropsychiatric disorders.

Disrupted extracellular matrix and cell cycle genes in autism-associated Shank3 deficiency are targeted by lithium.

The Shank3 gene encodes the major postsynaptic scaffolding protein SHANK3. Its mutation causes a syndromic form of autism spectrum disorder (ASD): Phelan-McDermid Syndrome (PMDS). It is characterized by global developmental delay, intellectual disorders (ID), ASD behavior, affective symptoms, as well as extra-cerebral symptoms. Although Shank3 deficiency causes a variety of molecular alterations, they do not suffice to explain all clinical aspects of this heterogenic syndrome. Since global gene expression alterations in Shank3 deficiency remain inadequately studied, we explored the transcriptome in vitro in primary hippocampal cells from Shank3∆11(-/-) mice, under control and lithium (Li) treatment conditions, and confirmed the findings in vivo. The Shank3∆11(-/-) genotype affected the overall transcriptome. Remarkably, extracellular matrix (ECM) and cell cycle transcriptional programs were disrupted. Accordingly, in the hippocampi of adolescent Shank3∆11(-/-) mice we found proteins of the collagen family and core cell cycle proteins downregulated. In vitro Li treatment of Shank3∆11(-/-) cells had a rescue-like effect on the ECM and cell cycle gene sets. Reversed ECM gene sets were part of a network, regulated by common transcription factors (TF) such as cAMP responsive element binding protein 1 (CREB1) and ß-Catenin (CTNNB1), which are known downstream effectors of synaptic activity and targets of Li. These TFs were less abundant and/or hypo-phosphorylated in hippocampi of Shank3∆11(-/-) mice and could be rescued with Li in vitro and in vivo. Our investigations suggest the ECM compartment and cell cycle genes as new players in the pathophysiology of Shank3 deficiency, and imply involvement of transcriptional regulators, which can be modulated by Li. This work supports Li as potential drug in the management of PMDS symptoms, where a Phase III study is ongoing.

Shank3 deletion in PV neurons is associated with abnormal behaviors and neuronal functions that are rescued by increasing GABAergic signaling.

BACKGROUND: Phelan-McDermid syndrome (PMS) is a neurodevelopmental disorder characterized by developmental delay, intellectual disability, and autistic-like behaviors and is primarily caused by haploinsufficiency of SHANK3 gene. Currently, there is no specific treatment for PMS, highlighting the need for a better understanding of SHANK3 functions and the underlying pathophysiological mechanisms in the brain. We hypothesize that SHANK3 haploinsufficiency may lead to alterations in the inhibitory system, which could be linked to the excitatory/inhibitory imbalance observed in models of autism spectrum disorder (ASD). Investigation of these neuropathological features may shed light on the pathogenesis of PMS and potential therapeutic interventions. METHODS: We recorded local field potentials and visual evoked responses in the visual cortex of Shank3∆11-/- mice. Then, to understand the impact of Shank3 in inhibitory neurons, we generated Pv-cre+/- Shank3Fl/Wt conditional mice, in which Shank3 was deleted in parvalbumin-positive neurons. We characterized the phenotype of this murine model and we compared this phenotype before and after ganaxolone administration. RESULTS: We found, in the primary visual cortex, an alteration of the gain control of Shank3 KO compared with Wt mice, indicating a deficit of inhibition on pyramidal neurons. This alteration was rescued after the potentiation of GABAA receptor activity by Midazolam. Behavioral analysis showed an impairment in grooming, memory, and motor coordination of Pv-cre+/- Shank3Fl/Wt compared with Pv-cre+/- Shank3Wt/Wt mice. These deficits were rescued with ganaxolone, a positive modulator of GABAA receptors. Furthermore, we demonstrated that treatment with ganaxolone also ameliorated evocative memory deficits and repetitive behavior of Shank3 KO mice. LIMITATIONS: Despite the significant findings of our study, some limitations remain. Firstly, the neurobiological mechanisms underlying the link between Shank3 deletion in PV neurons and behavioral alterations need further investigation. Additionally, the impact of Shank3 on other classes of inhibitory neurons requires further exploration. Finally, the pharmacological activity of ganaxolone needs further characterization to improve our understanding of its potential therapeutic effects. CONCLUSIONS: Our study provides evidence that Shank3 deletion leads to an alteration in inhibitory feedback on cortical pyramidal neurons, resulting in cortical hyperexcitability and ASD-like behavioral problems. Specifically, cell type-specific deletion of Shank3 in PV neurons was associated with these behavioral deficits. Our findings suggest that ganaxolone may be a potential pharmacological approach for treating PMS, as it was able to rescue the behavioral deficits in Shank3 KO mice. Overall, our study highlights the importance of investigating the role of inhibitory neurons and potential therapeutic interventions in neurodevelopmental disorders such as PMS.

Shank2/3 double knockout-based screening of cortical subregions links the retrosplenial area to the loss of social memory in autism spectrum disorders.

Members of the Shank protein family are master scaffolds of the postsynaptic architecture and mutations within the SHANK genes are causally associated with autism spectrum disorders (ASDs). We generated a Shank2-Shank3 double knockout mouse that is showing severe autism related core symptoms, as well as a broad spectrum of comorbidities. We exploited this animal model to identify cortical brain areas linked to specific autistic traits by locally deleting Shank2 and Shank3 simultaneously. Our screening of 10 cortical subregions revealed that a Shank2/3 deletion within the retrosplenial area severely impairs social memory, a core symptom of ASD. Notably, DREADD-mediated neuronal activation could rescue the social impairment triggered by Shank2/3 depletion. Data indicate that the retrosplenial area has to be added to the list of defined brain regions that contribute to the spectrum of behavioural alterations seen in ASDs.

SHANK3 Antibody Validation: Differential Performance in Western Blotting, Immunocyto- and Immunohistochemistry.

SHANK3 is a scaffolding protein implicated in autism spectrum disorders (ASD). Its function at excitatory glutamatergic synapses has been studied for the last two decades, however, tissue-specific expression patterns as well as its subcellular localization need to be studied in further detail. Especially the close sequence homology of SHANK3 to its protein family members SHANK2 and SHANK1 raises the emerging need for specific antibodies that are validated for the desired methodology. With this study, we aim to validate a set of commercial as well as homemade SHANK3 antibodies in Western Blotting, and synaptic immunocyto- and immunohistochemistry. We found that only a small subset of the antibodies included in this study meet the criteria of quality and specificity. Therefore, we aim to share our findings on SHANK3 antibody validation but also raise awareness of the necessity of antibody specificity testing in the field.

SHANK3 deficiency leads to myelin defects in the central and peripheral nervous system.

Mutations or deletions of the SHANK3 gene are causative for Phelan-McDermid syndrome (PMDS), a syndromic form of autism spectrum disorders (ASDs). We analyzed Shank3Δ11(-/-) mice and organoids from PMDS individuals to study effects on myelin. SHANK3 was found to be expressed in oligodendrocytes and Schwann cells, and MRI analysis of Shank3Δ11(-/-) mice revealed a reduced volume of the corpus callosum as seen in PMDS patients. Myelin proteins including myelin basic protein showed significant temporal and regional differences with lower levels in the CNS but increased amounts in the PNS of Shank3Δ11(-/-) animals. Node, as well as paranode, lengths were increased and ultrastructural analysis revealed region-specific alterations of the myelin sheaths. In PMDS hiPSC-derived cerebral organoids we observed an altered number and delayed maturation of myelinating cells. These findings provide evidence that, in addition to a synaptic deregulation, impairment of myelin might profoundly contribute to the clinical manifestation of SHANK3 deficiency.

Development of sex- and genotype-specific behavioral phenotypes in a Shank3 mouse model for neurodevelopmental disorders.

Individuals with a SHANK3-related neurodevelopmental disorder, also termed Phelan-McDermid syndrome or abbreviated as PMS, exhibit significant global developmental delay, language impairment, and muscular hypotonia. Also common are repetitive behaviors and altered social interactions, in line with a diagnosis of autism spectrum disorders. This study investigated the developmental aspect of autism-related behaviors and other phenotypes in a Shank3-transgenic mouse model. The animals underwent two sets of identical behavioral experiments, spanning motor skills, social and repetitive behavior, and cognition: baseline began at 5 weeks of age, corresponding to human adolescence, and the follow-up was initiated when aged 13 weeks, resembling early adulthood in humans. Interestingly, the animals displayed relatively stable phenotypes. Moreover, motor coordination and endurance were impaired, while muscle strength was unchanged. Surprisingly, the animals displayed only minor impairments in social behavior, but pronounced stereotypic and repetitive behaviors. Some behavioral tests indicated increased avoidance and anxiety. While spatial learning and memory were unchanged, knockout animals displayed slightly impaired cognitive flexibility. Female animals had similar abnormalities as males in the paradigms testing avoidance, anxiety, and cognition, but were less pathological in motor function and repetitive behavior. In all test paradigms, heterozygous Shank3 knockout animals had either no abnormal or a milder phenotype. Accurate characterization of animal models for genetic diseases is a prerequisite for understanding the pathophysiology. This is subsequently the basis for finding suitable and, ideally, translational biomarkers for therapeutic approaches and, thereby reducing the number of animals needed for preclinical trials.

Shank3 Transgenic and Prenatal Zinc-Deficient Autism Mouse Models Show Convergent and Individual Alterations of Brain Structures in MRI.

Research efforts over the past decades have unraveled both genetic and environmental factors, which contribute to the development of autism spectrum disorders (ASD). It is, to date, largely unknown how different underlying causes result in a common phenotype. However, the individual course of development and the different comorbidities might reflect the heterogeneous genetic and non-genetic contributions. Therefore, it is reasonable to identify commonalities and differences in models of these disorders at the different hierarchical levels of brain function, including genetics/environment, cellular/synaptic functions, brain regions, connectivity, and behavior. To that end, we investigated Shank3 transgenic mouse lines and compared them with a prenatal zinc-deficient (PZD) mouse model of ASD at the level of brain structural alterations in an 11,7 T small animal magnetic resonance imaging (MRI). Animals were measured at 4 and 9 weeks of age. We identified a decreased total brain volume (TBV) and hippocampal size of Shank3-/- mice but a convergent increase of basal ganglia (striatum and globus pallidus) in most mouse lines. Moreover, Shank3 transgenic mice had smaller thalami, whereas PZD mice had this region enlarged. Intriguingly, Shank3 heterozygous knockout mice mostly showed minor abnormalities to full knockouts, which might reflect the importance of proper Shank3 dosage in neuronal cells. Most reported volume changes seemed to be more pronounced at younger age. Our results indicate both convergent and divergent brain region abnormalities in genetic and non-genetic models of ASD. These alterations of brain structures might be mirrored in the reported behavior of both models, which have not been assessed in this study.

Blogging medical students: a qualitative analysis.

PURPOSE: Blogging is an increasingly popular method of sharing and reflecting on experiences of medical students in the World Wide Web with a potentially global learning community. The authors are not aware of studies that specifically examined blogs by medical students and thus for the first time investigated the type of experiences and impressions that emerged from these blogs with relevance for medical students and medical educators. METHOD: This was a qualitative study. Initially 75 blogs were identified. 33 blogs with a total of 1228 English and 337 German blog entries met the inclusion criteria and were analyzed. We started with line-by-line coding and switched to focused coding using constant comparative analysis to create a categorical framework for blogs. RESULTS: Medical students use blogs to write and reflect about a large variety of issues related to medical school. Major emerging themes included the preparation for written and oral high-stakes exams, experiences during clinical rotations, dealing with distressing situations during medical school, and social life of students beyond medical school. CONCLUSIONS: Our findings suggest that blogs are a potentially useful tool for medical students to reflect on their experiences during medical school as well as for medical educators to better understand how students perceive their time in medical school. The educational benefit of blogging might even be increased if trained medical educators would help to facilitate meaningful and targeted discussions emerging from blog entries and comment on students' learning challenges with the chance to reach a large community of learners.

CpG blocks immunosuppression by myeloid-derived suppressor cells in tumor-bearing mice.

PURPOSE: The Toll-like receptor (TLR) 9 ligand CpG has been used successfully for the immunotherapy of cancer. Chronic CpG application in tumor-free hosts leads, however, to the expansion of myeloid-derived suppressor cells (MDSC), which can cause T-cell suppression and may thus hamper the development of an effective immune response. Here, we investigated the effect of TLR9 activation on the function of MDSC in tumor-bearing mice. EXPERIMENTAL DESIGN: We investigated the effect of CpG treatment on the number, phenotype, and function of MDSC in mice bearing subcutaneous C26 tumors and in CEA424-TAg mice bearing autochthonous gastric tumors. RESULTS: CpG treatment blocks the suppressive activity of MDSC on T-cell proliferation in both tumor models. Inhibition of MDSC function by CpG was particularly pronounced for a highly suppressive Ly6G(hi) polymorphonuclear subset of MDSC. We further show that TLR9 activation by CpG promotes maturation and differentiation of MDSC and strongly decreases the proportion of Ly6G(hi) MDSC in both tumor-bearing and tumor-free mice. We demonstrate that IFN-α produced by plasmacytoid dendritic cells upon CpG stimulation is a key effector for the induction of MDSC maturation in vitro and show that treatment of mice with recombinant IFN-α is sufficient to block MDSC suppressivity. CONCLUSIONS: We show here for the first time that TLR9 activation inhibits the regulatory function of MDSC in tumor-bearing mice and define a role for the antitumoral cytokine IFN-α in this process.