Conference Agenda

Copy number variations (CNVs) at the chromosomal region 16p11.2 are associated with autism spectrum disorder, intellectual disability and other neurodevelopmental disorders (NDDs).

Converging evidence from mouse models and human studies points to MAPK3, a gene located in the 16p11.2 region, as a key factor for NDD. MAPK3 encodes for ERK1, a protein kinase of ERK signalling cascade, which regulates neurodevelopment, cognition and behavioural plasticity. Moreover, recent data suggest that dysfunctions in the cortico-striatal connectivity underlie NDDs in both patients affected by NDDs and mouse models of 16p11.2 deletion.

To better elucidate the role of ERK signalling in the pathophysiology of NDDs associated with 16p11.2 CNVs, we employed both mouse models of 16p11.2 CNVs and human iPSCs-derived neurons. In addition, human carriers of 16p11.2 deletion and duplication were screened for peripheral alterations of ERK signalling components.

Our data demonstrate that MAPK3/ERK1 genetic alterations are reflected in corresponding biochemical changes in blood samples human 16p11.2 deletion and duplication patients.

In addition, converging evidence from our mouse models and iPSCs-derived neurons points to a potential dysregulation of dopaminergic signalling in 16p11.2 CNVs.

Altogether, our results suggest that peripheral signalling intermediates may become reliable biomarkers for NDDs. Moreover, the 16p11.2 CNVs may be implicated in cortico-striatal dysfunctions underlying NDDs.

Mounting evidence indicate that developmental deficits in cell proliferation and synaptic formation are shared signatures among neurodevelopmental disorders (NDs), including autism-spectrum disorders (ASD). It has been suggested that pleiotropic signal transduction pathways modulating processes such as transcription, translation, synaptic transmission and plasticity, epigenetics and immunoinflammatory responses, are involved in these pathological processes. Despite this new knowledge, we are currently unable to fully understand the molecular and cellular differences that underlie the clinical heterogeneity displayed by different forms of NDs/ASDs, a challenge made harder by the extreme complexity of these signaling pathways that quite often interact to produce the pathophysiological signs. Such complication has also hindered the identification of efficient biomarkers to assess both disease progression and therapeutic outcomes, an urgent clinical need. Here, I will highlight our recent advances on the comprehension of the molecular, cellular, and extracellular processes that are related to the occurrence, development, and outcome of CDKL5-deficiency disorder (CDD), a severe form of syndromic ASD caused by mutations in the X-linked Cyclin-dependent kinase-like 5 gene. CDKL5 is a serine/threonine kinase that is expressed early during postnatal development in neurons where it phosphorylates epigenetic factors, synaptic elements of both axonal and dendritic compartment and microtubule-associated proteins. By comparing functional, anatomical, and molecular scars left in the cerebral cortex by either total or cell-type specific mutation of CDKL5, we found that neuronal and glial organization as well as synaptic connectivity share common alterations among CDD mouse models and post-mortem samples from CDD patients. Moreover, parallel studies are revealing that loss of CDKL5 expression can affect neuronal responses via extracellular vesicles-mediated intercellular transfer of information. Overall, our results have revealed that CDD show deficits in pathways highly convergent with other NDs, strengthening the possibility of leveraging on these data to better illuminate their pathophysiology.

Abnormal tactile response is considered an integral feature of Autism Spectrum Disorders (ASDs), and hypo-responsiveness to tactile stimuli is often associated with the severity of ASDs core symptoms. Mutations in the human CNTNAP2 and SHANK3 genes result in cortical dysplasia-focal epilepsy syndrome (CDFE) and Phelan-McDermid syndrome (PMS) respectively, two syndromic forms of autism. Likewise, Cntnap2-/- and Shank3b-/- mice show deficits relevant to core symptoms of human ASDs. Sensory abnormalities have been described in mice lacking ASD-associated genes. However, the neural underpinnings of these somatosensory abnormalities are still poorly understood. Here we investigated, in Cntnap2-/- and Shank3b-/- mice, the neural substrates of whisker-mediated responses, a key component of rodents’ interaction with the surrounding environment.
When compared to their controls, both Cntnap2-/- and Shank3b-/- mice displayed impaired whisker-dependent discrimination in the textured novel object recognition test. Additionally, Shank3b-/- but not Cntnap2-/- mice showed a marked behavioural hypo-responsiveness to repetitive whisker stimulation in the whisker nuisance test. Notably, while Cntnap2-/- mice displayed increased c-fos mRNA induction within primary somatosensory cortex (S1) following whisker stimulation, Shank3b-/- mice showed a significantly reduced activation of S1. Moreover, when tested in a resting-state fMRI paradigm, Cntnap2-/- mice showed focal hyper-connectivity within the S1, while reduced S1-hippocampal connectivity was found in Shank3b-/- mice.
Together, these findings suggest that impaired neuronal activation and dysfunctional connectivity within S1 might underlie hypo-reactivity to whisker-dependent cues in Cntnap2-/- and Shank3b-/- mice, highlighting a potentially generalizable form of dysfunctional somatosensory processing in ASD.

The neural stem cell niche has a crucial role in determining cell fate and maturation, however its importance is often overlooked. In vitro brain organoids attempt to mimic the organ but achieve only partial recapitulation. In this work, we investigated whether the temporal regulation of tissue-specific growth factors and the presence of a biomimetic extracellular matrix (ECM) could influence the potential of embryonic rat neural stem cells to generate hippocampal organoids. To this aim, we compared three protocols, here named SiFa, with BDNF used as a single differentiation factor, MuFa where multiple factors responsible for hippocampal neurogenesis, such as WNT, SHH, BMP or their agonists were added besides BDNF, and MuFaM. The latter consisted of the MuFa protocol supplemented with an alginate-based ECM. The organoids grown in all three protocols showed the early formation of the dorsal-ventral axis, as noticed by the polarized localization of the ventral marker Pax6. In MuFaM organoids we observed an increased number of mature MAP2+ neurons, compared to when using growth factors alone. Moreover, the biomimetic ECM decreased the stiffness of the organoids while increasing their adhesion, suggesting a possible link between the organoid physicomechanical properties and neuronal differentiation. On the other hand, MuFa organoids showed a specific regionalization of GFAP+ cells sandwiched between two layers of Tubb3+ cells. The native ECM markers Laminin and Tenascin R showed a different patterning across the conditions. In particular, the two ECM markers localized in two separate layers on one side only of MuFa organoids, showing a similarity to their specific localization in the ventral hippocampus of postnatal rats. In conclusion, different microenvironment factors influenced differently the cytoarchitecture, ECM distribution and neuron maturation of hippocampal organoids. These findings provide insight into the complexity of the different factors to consider for recapitulating brain structures in vitro.