Trans-cardiac perfusion of neonatal mice and immunofluorescence of the whole body as a method to study nervous system development.

Whole animal perfusion is a well-established method that has been used for the past decades in multiple research fields. Particularly, it has been very important for the study of the brain. The rapid and uniform fixation of tissue is essential for the preservation of its integrity and the study of complex structures. For small tissue pieces submerging in formaldehyde solution oftentimes is sufficient to get a good fixation, larger tissues or organs with a more complicated structure present a greater difficulty. Here, we report the precise parameters to successfully perform trans-cardiac perfusion of neonatal mouse pups that allows a uniform fixation of the whole body for subsequent structural analysis and immunohistochemistry. In comparison to standard perfusion procedures of adult mice, changes in the pump velocity, the buffer volume and in the needle size lead to high quality fixation of neonatal mice pups. Further, we present a whole-body section staining, which results in a highly specific immunofluorescence signal suited for detailed analysis of multiple tissues or systems at the same time. Thus, our protocol provides a reproducible and reliable method for neonatal perfusion and staining that can rapidly be applied in any laboratory. It allows a high quality analysis of cellular structures and expression profiles at early developmental stages.

The ADORA1 mutation linked to early-onset Parkinson's disease alters adenosine A1-A2A receptor heteromer formation and function.

Adenosine modulates neurotransmission through inhibitory adenosine A1 receptors (A1Rs) and stimulatory A2A receptors (A2ARs). These G protein-coupled receptors are involved in motor function and related to neurodegenerative diseases such as Parkinson's disease (PD). An autosomal-recessive mutation (G2797.44S) within the transmembrane helix (TM) 7 of A1R (A1RG279S) has been associated with the development of early onset PD (EOPD). Here, we aimed at investigating the impact of this mutation on the structure and function of the A1R and the A1R-A2AR heteromer. Our results revealed that the G2797.44S mutation does not alter A1R expression, ligand binding, constitutive activity or coupling to transducer proteins (Gαi, Gαq, Gα12/13, Gαs, ß-arrestin2 and GRK2) in transfected HEK-293 T cells. However, A1RG279S weakened the ability of A1R to heteromerize with A2AR, as shown in a NanoBiT assay, which led to the disappearance of the heteromerization-dependent negative allosteric modulation that A1R imposes on the constitutive activity and agonist-induced activation of the A2AR. Molecular dynamic simulations allowed to propose an indirect mechanism by which the G2797.44S mutation in TM 7 of A1R weakens the TM 5/6 interface of the A1R-A2AR heteromer. Therefore, it is demonstrated that a PD linked ADORA1 mutation is associated with dysfunction of adenosine receptor heteromerization. We postulate that a hyperglutamatergic state secondary to increased constitutive activity and sensitivity to adenosine of A2AR not forming heteromers with A1R could represent a main pathogenetic mechanism of the EOPD associated with the G2797.44S ADORA1 mutation.

SHANK2 Mutations Result in Dysregulation of the ERK1/2 Pathway in Human Induced Pluripotent Stem Cells-Derived Neurons and Shank2(-/-) Mice.

SHANK2 (ProSAP1) is a postsynaptic scaffolding protein of excitatory synapses in the central nervous system and implicated in the development of autism spectrum disorders (ASD). Patients with mutations in SHANK2 show autism-like behaviors, developmental delay, and intellectual disability. We generated human induced pluripotent stem cells (hiPSC) from a patient carrying a heterozygous deletion of SHANK2 and from the unaffected parents. In patient hiPSCs and derived neurons SHANK2 mRNA and protein expression was reduced. During neuronal maturation, a reduction in growth cone size and a transient increase in neuronal soma size were observed. Neuronal proliferation was increased, and apoptosis was decreased in young and mature neurons. Additionally, mature patient hiPSC-derived neurons showed dysregulated excitatory signaling and a decrease of a broad range of signaling molecules of the ERK-MAP kinase pathway. These findings could be confirmed in brain samples from Shank2(-/-) mice, which also showed decreased mGluR5 and phospho-ERK1/2 expression. Our study broadens the current knowledge of SHANK2-related ASD. We highlight the importance of excitatory-inhibitory balance and mGluR5 dysregulation with disturbed downstream ERK1/2 signaling in ASD, which provides possible future therapeutic strategies for SHANK2-related ASD.