Transcranial two-photon imaging of synaptic structures in the cortex of awake head-restrained mice.

Transcranial two-photon microscopy allows long-term imaging of neurons, glia, and vasculature in the intact cortex of living animals. So far, this technique has been primarily used to acquire images in anesthetized animals. Here, we describe a detailed protocol for high-resolution two-photon imaging of neuronal structures in the cortex of awake head-restrained mice. Surgery is done within 1 h in anesthetized mice. After animals recover from anesthesia, two-photon imaging can be performed multiple times over minutes to days, allowing longitudinal studies of synaptic plasticity and pathology without the complication induced by anesthesia reagents.

Lis1 controls dynamics of neuronal filopodia and spines to impact synaptogenesis and social behaviour.

LIS1 (PAFAH1B1) mutation can impair neuronal migration, causing lissencephaly in humans. LIS1 loss is associated with dynein protein motor dysfunction, and disrupts the actin cytoskeleton through disregulated RhoGTPases. Recently, LIS1 was implicated as an important protein-network interaction node with high-risk autism spectrum disorder genes expressed in the synapse. How LIS1 might participate in this disorder has not been investigated. We examined the role of LIS1 in synaptogenesis of post-migrational neurons and social behaviour in mice. Two-photon imaging of actin-rich dendritic filopodia and spines in vivo showed significant reductions in elimination and turnover rates of dendritic protrusions of layer V pyramidal neurons in adolescent Lis1(+/-) mice. Lis1(+/-) filopodia on immature hippocampal neurons in vitro exhibited reduced density, length and RhoA dependent impaired dynamics compared to Lis1(+/+) . Moreover, Lis1(+/-) adolescent mice exhibited deficits in social interaction. Lis1 inactivation restricted to the postnatal hippocampus resulted in similar deficits in dendritic protrusion density and social interactions. Thus, LIS1 plays prominently in dendritic filopodia dynamics and spine turnover implicating reduced dendritic spine plasticity as contributing to developmental autistic-like behaviour.

Constitutive activation of signal transducers and activators of transcription predicts vorinostat resistance in cutaneous T-cell lymphoma.

Vorinostat is a histone deacetylase inhibitor that induces differentiation, growth arrest, and/or apoptosis of malignant cells both in vitro and in vivo and has shown clinical responses in approximately 30% of patients with advanced mycosis fungoides and Sézary syndrome cutaneous T-cell lymphoma (CTCL). The purpose of this study was to identify biomarkers predictive of vorinostat response in CTCL using preclinical model systems and to assess these biomarkers in clinical samples. The signal transducer and activator of transcription (STAT) signaling pathway was evaluated. The data indicate that persistent activation of STAT1, STAT3, and STAT5 correlate with resistance to vorinostat in lymphoma cell lines. Simultaneous treatment with a pan-Janus-activated kinase inhibitor resulted in synergistic antiproliferative effect and down-regulation of the expression of several antiapoptotic genes. Immunohistochemical analysis of STAT1 and phosphorylated tyrosine STAT3 (pSTAT3) in skin biopsies obtained from CTCL patients enrolled in the vorinostat phase IIb trial showed that nuclear accumulation of STAT1 and high levels of nuclear pSTAT3 in malignant T cells correlate with a lack of clinical response. These results suggest that deregulation of STAT activity plays a role in vorinostat resistance in CTCL, and strategies that block this pathway may improve vorinostat response. Furthermore, these findings may be of prognostic value in predicting the response of CTCL patients to vorinostat.