Expression of Dystrophin Dp71 Splice Variants Is Temporally Regulated During Rodent Brain Development.

Dystrophin Dp71 is the major product of the Duchenne muscular dystrophy (DMD) gene in the brain, and its loss in DMD patients and mouse models leads to cognitive impairments. Dp71 is expressed as a range of proteins generated by alternative splicing of exons 71 to 74 and 78, classified in the main Dp71d and Dp71f groups that contain specific C-terminal ends. However, it is unknown whether each isoform has a specific role in distinct cell types, brain regions, and/or stages of brain development. In the present study, we characterized the expression of Dp71 isoforms during fetal (E10.5, E15.5) and postnatal (P1, P7, P14, P21 and P60) mouse and rat brain development. We finely quantified the expression of several Dp71 transcripts by RT-PCR and cloning assays in samples from whole-brain and distinct brain structures. The following Dp71 transcripts were detected: Dp71d, Dp71d∆71, Dp71d∆74, Dp71d∆71,74, Dp71d∆71-74, Dp71f, Dp71f∆71, Dp71f∆74, Dp71f∆71,74, and Dp71fΔ71-74. We found that the Dp71f isoform is the main transcript expressed at E10.5 (> 80%), while its expression is then progressively reduced and replaced by the expression of isoforms of the Dp71d group from E15.5 to postnatal and adult ages. This major finding was confirmed by third-generation nanopore sequencing. In addition, we found that the level of expression of specific Dp71 isoforms varies as a function of postnatal stages and brain structure. Our results suggest that Dp71 isoforms have different and complementary roles during embryonic and postnatal brain development, likely taking part in a variety of maturation processes in distinct cell types.

Pharmacological Inhibition of p-21 Activated Kinase (PAK) Restores Impaired Neurite Outgrowth and Remodeling in a Cellular Model of Down Syndrome.

Down syndrome (DS) is characterized by the trisomy of chromosome 21 and by cognitive deficits that have been related to neuronal morphological alterations in humans, as well as in animal models. The gene encoding for amyloid precursor protein (APP) is present in autosome 21, and its overexpression in DS has been linked to neuronal dysfunction, cognitive deficit, and Alzheimer's disease-like dementia. In particular, the neuronal ability to extend processes and branching is affected. Current evidence suggests that APP could also regulate neurite growth through its role in the actin cytoskeleton, in part by influencing p21-activated kinase (PAK) activity. The latter effect is carried out by an increased abundance of the caspase cleavage-released carboxy-terminal C31 fragment. In this work, using a neuronal cell line named CTb, which derived from the cerebral cortex of a trisomy 16 mouse, an animal model of human DS, we observed an overexpression of APP, elevated caspase activity, augmented cleavage of the C-terminal fragment of APP, and increased PAK1 phosphorylation. Morphometric analyses showed that inhibition of PAK1 activity with FRAX486 increased the average length of the neurites, the number of crossings per Sholl ring, the formation of new processes, and stimulated the loss of processes. Considering our results, we propose that PAK hyperphosphorylation impairs neurite outgrowth and remodeling in the cellular model of DS, and therefore we suggest that PAK1 may be a potential pharmacological target.

Overexpressed Down Syndrome Cell Adhesion Molecule (DSCAM) Deregulates P21-Activated Kinase (PAK) Activity in an In Vitro Neuronal Model of Down Syndrome: Consequences on Cell Process Formation and Extension.

In humans, Down syndrome (DS) is caused by the presence of an extra copy of autosome 21. The most striking finding in DS patients is intellectual disability and the onset of Alzheimer's disease (AD)-like neuropathology in adulthood. Gene overdose is most likely to underlie both developmental impairments, as well as altered neuronal function in DS. Lately, the disruption of cellular signaling and regulatory pathways has been implicated in DS pathophysiology, and many of such pathways may represent common targets for diverse DS-related genes, which could in turn represent attractive therapeutical targets. In this regard, one DS-related gene Down Syndrome Cell Adhesion Molecule (DSCAM), has important functions in neuronal proliferation, maturation, and synaptogenesis. p21-associated kinases (PAKs) appear as a most interesting possibility for study, as DSCAM is known to regulate the PAKs pathway. Hence, in DS, overexpressed DSCAM could deregulate PAKs activity and affect signaling pathways that regulate synaptic plasticity such as dendritic spine dynamics and axon guidance and growth. In the present work, we used an immortalized cell line derived from the cerebral cortex of an animal model of DS such as the trisomy 16 (Ts16) fetal mouse (named CTb), and a similar cell line established from a normal littermate (named CNh), to study the effect of DSCAM in the PAKs pathway. The present study shows that DSCAM is overexpressed in CTb cells by approximately twofold, compared to CNh cells. Congruently, PAK1, as well as its downstream effectors LIMK and cofilin, stay phosphorylated for longer periods after DSCAM activation in the CTb cells, leading to an altered actin dynamics, expressed as an increased basal F/G ratio and reduced neurite growth, in the trisomic condition. The present work presents the correlation between DSCAM gene overexpression and a dysregulation of the PAK pathway, resulting in altered morphological parameters of neuronal plasticity in the trisomic cell line, namely decreased number and length of processes.