ßII-Spectrin Is Required for Synaptic Positioning during Retinal Development.

Neural circuit assembly is a multistep process where synaptic partners are often born at distinct developmental stages, and yet they must find each other and form precise synaptic connections with one another. This developmental process often relies on late-born neurons extending their processes to the appropriate layer to find and make synaptic connections to their early-born targets. The molecular mechanism responsible for the integration of late-born neurons into an emerging neural circuit remains unclear. Here, we uncovered a new role for the cytoskeletal protein ßII-spectrin in properly positioning presynaptic and postsynaptic neurons to the developing synaptic layer. Loss of ßII-spectrin disrupts retinal lamination, leads to synaptic connectivity defects, and results in impaired visual function in both male and female mice. Together, these findings highlight a new function of ßII-spectrin in assembling neural circuits in the mouse outer retina.SIGNIFICANCE STATEMENT Neurons that assemble into a functional circuit are often integrated at different developmental time points. However, the molecular mechanism that guides the precise positioning of neuronal processes to the correct layer for synapse formation is relatively unknown. Here, we show a new role for the cytoskeletal scaffolding protein, ßII-spectrin in the developing retina. ßII-spectrin is required to position presynaptic and postsynaptic neurons to the nascent synaptic layer in the mouse outer retina. Loss of ßII-spectrin disrupts positioning of neuronal processes, alters synaptic connectivity, and impairs visual function.

Neurofascin Is a Novel Component of Rod Photoreceptor Synapses in the Outer Retina.

Neural circuit formation is an intricate and complex process where multiple neuron types must come together to form synaptic connections at a precise location and time. How this process is orchestrated during development remains poorly understood. Cell adhesion molecules are known to play a pivotal role in assembling neural circuits. They serve as recognition molecules between corresponding synaptic partners. In this study, we identified a new player in assembling neural circuits in the outer retina, the L1-family cell adhesion molecule Neurofascin (Nfasc). Our data reveals Nfasc is expressed in the synaptic layer where photoreceptors make synaptic connections to their respective partners. A closer examination of Nfasc expression shows high levels of expression in rod bipolars but not in cone bipolars. Disruption of Nfasc using a conditional knockout allele results in selective loss of pre- and post-synaptic proteins in the rod synaptic layer but not in the cone synaptic layer. Electron microscopic analysis confirms that indeed there are abnormal synaptic structures with less dendrites of rod bipolars innervating rod terminals in loss of Nfasc animals. Consistent with these findings, we also observe a decrease in rod-driven retinal responses with disruption of Nfasc function but not in cone-driven responses. Taken together, our data suggest a new role of Nfasc in rod synapses within the mouse outer retina.

PMN-MDSCs Enhance CTC Metastatic Properties through Reciprocal Interactions via ROS/Notch/Nodal Signaling.

Intratumoral infiltration of myeloid-derived suppressor cells (MDSCs) is known to promote neoplastic growth by inhibiting the tumoricidal activity of T cells. However, direct interactions between patient-derived MDSCs and circulating tumors cells (CTCs) within the microenvironment of blood remain unexplored. Dissecting interplays between CTCs and circulatory MDSCs by heterotypic CTC/MDSC clustering is critical as a key mechanism to promote CTC survival and sustain the metastatic process. We characterized CTCs and polymorphonuclear-MDSCs (PMN-MDSCs) isolated in parallel from peripheral blood of metastatic melanoma and breast cancer patients by multi-parametric flow cytometry. Transplantation of both cell populations in the systemic circulation of mice revealed significantly enhanced dissemination and metastasis in mice co-injected with CTCs and PMN-MDSCs compared to mice injected with CTCs or MDSCs alone. Notably, CTC/PMN-MDSC clusters were detected in vitro and in vivo either in patients' blood or by longitudinal monitoring of blood from animals. This was coupled with in vitro co-culturing of cell populations, demonstrating that CTCs formed physical clusters with PMN-MDSCs; and induced their pro-tumorigenic differentiation through paracrine Nodal signaling, augmenting the production of reactive oxygen species (ROS) by PMN-MDSCs. These findings were validated by detecting significantly higher Nodal and ROS levels in blood of cancer patients in the presence of naïve, heterotypic CTC/PMN-MDSC clusters. Augmented PMN-MDSC ROS upregulated Notch1 receptor expression in CTCs through the ROS-NRF2-ARE axis, thus priming CTCs to respond to ligand-mediated (Jagged1) Notch activation. Jagged1-expressing PMN-MDSCs contributed to enhanced Notch activation in CTCs by engagement of Notch1 receptor. The reciprocity of CTC/PMN-MDSC bi-directional paracrine interactions and signaling was functionally validated in inhibitor-based analyses, demonstrating that combined Nodal and ROS inhibition abrogated CTC/PMN-MDSC interactions and led to a reduction of CTC survival and proliferation. This study provides seminal evidence showing that PMN-MDSCs, additive to their immuno-suppressive roles, directly interact with CTCs and promote their dissemination and metastatic potency. Targeting CTC/PMN-MDSC heterotypic clusters and associated crosstalks can therefore represent a novel therapeutic avenue for limiting hematogenous spread of metastatic disease.

Targeting USP7 Identifies a Metastasis-Competent State within Bone Marrow-Resident Melanoma CTCs.

Systemic metastasis is the major cause of death from melanoma, the most lethal form of skin cancer. Although most patients with melanoma exhibit a substantial gap between onset of primary and metastatic tumors, signaling mechanisms implicated in the period of metastatic latency remain unclear. We hypothesized that melanoma circulating tumor cells (CTC) home to and reside in the bone marrow during the asymptomatic phase of disease progression. Using a strategy to deplete normal cell lineages (Lin-), we isolated CTC-enriched cell populations from the blood of patients with metastatic melanoma, verified by the presence of putative CTCs characterized by melanoma-specific biomarkers and upregulated gene transcripts involved in cell survival and prodevelopment functions. Implantation of Lin- population in NSG mice (CTC-derived xenografts, i.e., CDX), and subsequent transcriptomic analysis of ex vivo bone marrow-resident tumor cells (BMRTC) versus CTC identified protein ubiquitination as a significant regulatory pathway of BMRTC signaling. Selective inhibition of USP7, a key deubiquinating enzyme, arrested BMRTCs in bone marrow locales and decreased systemic micrometastasis. This study provides first-time evidence that the asymptomatic progression of metastatic melanoma can be recapitulated in vivo using patient-isolated CTCs. Furthermore, these results suggest that USP7 inhibitors warrant further investigation as a strategy to prevent progression to overt clinical metastasis.Significance: These findings provide insights into mechanism of melanoma recurrence and propose a novel approach to inhibit systematic metastatic disease by targeting bone marrow-resident tumor cells through pharmacological inhibition of USP7.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/18/5349/F1.large.jpg Cancer Res; 78(18); 5349-62. ©2018 AACR.