Lina Chakrabarti

Defects in Embryonic Neurogenesis and Initial Synapse Formation in the Forebrain of the Ts65Dn Mouse Model of Down Syndrome

Trisomy 21, one of the most prevalent congenital birth defects, results in a constellation of phenotypes collectively termed Down syndrome (DS). Mental retardation and motor and sensory deficits are among the many debilitating symptoms of DS. Alterations in brain growth and synaptic development are thought to underlie the cognitive impairments in DS, but the role of early brain development has not been studied because of the lack of embryonic human tissue and because of breeding difficulties in mouse models of DS. We generated a breeding colony of the Ts65Dn mouse model of DS to test the hypothesis that early defects in embryonic brain development are a component of brain dysfunction in DS. We found substantial delays in prenatal growth of the Ts65Dn cerebral cortex and hippocampus because of longer cell cycle duration and reduced neurogenesis from the ventricular zone neural precursor population. In addition, the Ts65Dn neocortex remains hypocellular after birth and there is a lasting decrease in synaptic development beginning in the first postnatal week. These results demonstrate that specific abnormalities in embryonic forebrain precursor cells precede early deficits in synaptogenesis and may underlie the postnatal disabilities in Ts65Dn and DS. The early prenatal period is therefore an important new window for possible therapeutic amelioration of the cognitive symptoms in DS.

Olig1 and Olig2 triplication causes developmental brain defects in Down syndrome

Over-inhibition is thought to be one of the underlying causes of the cognitive deficits in Ts65Dn mice, the most widely used model of Down syndrome. We found a direct link between gene triplication and defects in neuron production during embryonic development. These neurogenesis defects led to an imbalance between excitatory and inhibitory neurons and to increased inhibitory drive in the Ts65Dn forebrain. We discovered that Olig1 and Olig2, two genes that are triplicated in Down syndrome and in Ts65Dn mice, were overexpressed in the Ts65Dn forebrain. To test the hypothesis that Olig triplication causes the neurological phenotype, we used a genetic approach to normalize the dosage of these two genes and thereby rescued the inhibitory neuron phenotype in the Ts65Dn brain. These data identify seminal alterations during brain development and suggest a mechanistic relationship between triplicated genes and these brain abnormalities in the Ts65Dn mouse.

In vivo quantum dot labeling of mammalian stem and progenitor cells

Fluorescent semiconductor nanocrystal quantum dots (QDs) are a class of multifunctional inorganic fluorophores that hold great promise for clinical applications and biomedical research. Because no methods currently exist for directed QD‐labeling of mammalian cells in the nervous system in vivo, we developed novel in utero electroporation and ultrasound‐guided in vivo delivery techniques to efficiently and directly label neural stem and progenitor cells (NSPCs) of the developing mammalian central nervous system with QDs. Our initial safety and proof of concept studies of one and two‐cell QD‐labeled mouse embryos reveal that QDs are compatible with early mammalian embryonic development. Our in vivo experiments further show that in utero labeled NSPCs continue to develop in an apparent normal manner. These studies reveal that QDs can be effectively used to label mammalian NSPCs in vivo and will be useful for studies of in vivo fate mapping, cellular migration, and NSPC differentiation during mammalian development. Developmental Dynamics 236:3393–3401, 2007.

Environmental Enrichment Rescues Postnatal Neurogenesis Defect in the Male and Female Ts65Dn Mouse Model of Down Syndrome

Down syndrome (DS), the most frequent genetic cause of intellectual disability and developmental delay, results from impaired neural stem cell proliferation and differentiation. Impaired neurogenesis in the neocortex, hippocampus and cerebellum is believed to be the underlying cause of learning and behavioral deficits in the Ts65Dn mouse model of DS. Aggressive sensorimotor and cognitive therapies have shown promise in mitigating the cognitive disabilities in DS but these behavioral therapies have not yet been investigated at the cellular level. Here, using the Ts65Dn mouse model of DS, we demonstrate that a combination of environmental enrichment and physical exercise starting in juvenile mice (postnatal day 18) markedly increases cell proliferation, neurogenesis and gliogenesis in the hippocampal dentate gyrus (DG) and the forebrain subventricular zone (SVZ) of both male and female mice. Enrichment and exercise increased the rate of Ts65Dn DG neurogenesis to be comparable to that of the nonenriched euploid group, while the effect on SVZ neurogenesis was reduced and seen only after prolonged exposure. These results clearly indicate that in a comprehensive stimulatory environment, the postnatal DS brain has the intrinsic capability of improving neurogenesis and gliogenesis to the levels of normal matched controls and that this cellular response underlies the cognitive improvement seen following behavioral therapies.

Elevated Expression of the G-Protein-Activated Inwardly Rectifying Potassium Channel 2 (GIRK2) in Cerebellar Unipolar Brush Cells of a Down Syndrome Mouse Model

SUMMARY1. Down syndrome (DS) arises from the presence of three copies of chromosome (Chr.) 21. Fine motor learning deficits found in DS from childhood to adulthood result from expression of extra genes on Chr. 21, however, it remains unclear which if any of these genes are the specific causes of the cognitive and motor dysfunction. DS cerebellum displays morphological abnormalities that likely contribute to the DS motor phenotype.2. The G-protein-activated inwardly rectifying potassium channel subunit 2 (GIRK2) is expressed in cerebellum and can shunt dendritic conductance and attenuate postsynaptic potentials. We have used an interbreeding approach to cross a genetic mouse model of DS (Ts65Dn) with Girk2 knockout mice and examined its relative expression level by quantitative real-time RT-PCR, Western blotting and immunohistochemistry.3. We report here for the first time that GIRK2 is expressed in unipolar brush cells, which are excitatory interneurons of the vestibulocerebellum and dorsal cochlear nucleus. Analysis of disomic-Ts65Dn/Girk2(+/+/−) and heterozygous-Diploid/Girk2(+/−) mice shows that GIRK2 expression in Ts65Dn lobule X follows gene dosage. The lobule X of Ts65Dn mice contain greater numbers of unipolar brush cells co-expressing GIRK2 and calretinin than the control mouse groups.4. These results demonstrate that gene triplication can impact specific cell types in the cerebellum. We hypothesize that GIRK2 overexpression will adversely affect cerebellar circuitry in Ts65Dn vestibulocerebellum and dorsal cochlear nucleus due to GIRK2 shunting properties and its effects on resting membrane potential.

Prussian blue nanoparticles for laser-induced photothermal therapy of tumors

We describe Prussian blue nanoparticles (PB NPs) for laser-induced photothermal therapy (PTT) of tumors. The PB NPs exhibit strong absorbance at near infrared (NIR) wavelengths, are stable, non-toxic, and have 20.5% photothermal conversion efficiencies. PTT with PB NPs in a mouse model of neuroblastoma resulted in marked tumor debulking, increased tumor-free days, and decreased tumor growth rates in tumor-bearing mice. These findings demonstrate the clinical potential of PB NPs for PTT of tumors.

Reversible Adaptive Plasticity: A Mechanism for Neuroblastoma Cell Heterogeneity and Chemo-Resistance

We describe a novel form of tumor cell plasticity characterized by reversible adaptive plasticity in murine and human neuroblastoma. Two cellular phenotypes were defined by their ability to exhibit adhered, anchorage dependent (AD) or sphere forming, anchorage independent (AI) growth. The tumor cells could transition back and forth between the two phenotypes and the transition was dependent on the culture conditions. Both cell phenotypes exhibited stem-like features such as expression of nestin, self-renewal capacity, and mesenchymal differentiation potential. The AI tumorspheres were found to be more resistant to chemotherapy and proliferated slower in vitro compared to the AD cells. Identification of specific molecular markers like MAP2, β-catenin, and PDGFRβ enabled us to characterize and observe both phenotypes in established mouse tumors. Irrespective of the phenotype originally implanted in mice, tumors grown in vivo show phenotypic heterogeneity in molecular marker signatures and are indistinguishable in growth or histologic appearance. Similar molecular marker heterogeneity was demonstrated in primary human tumor specimens. Chemotherapy or growth factor receptor inhibition slowed tumor growth in mice and promoted initial loss of AD or AI heterogeneity, respectively. Simultaneous targeting of both phenotypes led to further tumor growth delay with emergence of new unique phenotypes. Our results demonstrate that neuroblastoma cells are plastic, dynamic, and may optimize their ability to survive by changing their phenotype. Phenotypic switching appears to be an adaptive mechanism to unfavorable selection pressure and could explain the phenotypic and functional heterogeneity of neuroblastoma.

A mechanism linking Id2-TGFβ crosstalk to reversible adaptive plasticity in neuroblastoma

The ability of high-risk neuroblastoma to survive unfavorable growth conditions and multimodal therapy has produced an elusive childhood cancer with remarkably poor prognosis. A novel phenomenon enabling neuroblastoma to survive selection pressure is its capacity for reversible adaptive plasticity. This plasticity allows cells to transition between highly proliferative anchorage dependent (AD) and slow growing, anoikis-resistant anchorage independent (AI) phenotypes. Both phenotypes are present in established mouse and human tumors. The differential gene expression profile of the two cellular phenotypes in the mouse Neuro2a cell line delineated pathways of proliferation in AD cells or tyrosine kinase activation/ apoptosis inhibition in AI cells. A 20 fold overexpression of inhibitor of differentiation 2 (Id2) was identified in AD cells while up-regulation of genes involved in anoikis resistance like PI3K/Akt, Erk, Bcl2 and integrins was observed in AI cells. Similarly, differential expression of Id2 and other genes of interest were also observed in the AD and AI phenotypes of human neuroblastoma cell lines, SK-N-SH and IMR-32; as well as in primary human tumor specimens. Forced down-regulation of Id2 in AD cells or overexpression in AI cells induced the cells to gain characteristics of the other phenotype. Id2 binds both TGFβ and Smad2/3 and appears critical for maintaining the proliferative phenotype at least partially through negative regulation of the TGFβ/Smad pathway. Simultaneously targeting the differential molecular pathways governing reversible adaptive plasticity resulted in 50% cure of microscopic disease and delayed tumor growth in established mouse neuroblastoma tumors. We present a mechanism that accounts for reversible adaptive plasticity and a molecular basis for combined targeted therapies in neuroblastoma.

Combination of Id2 Knockdown Whole Tumor Cells and Checkpoint Blockade: A Potent Vaccine Strategy in a Mouse Neuroblastoma Model.

Tumor vaccines have held much promise, but to date have demonstrated little clinical success. This lack of success is conceivably due to poor tumor antigen presentation combined with immuno-suppressive mechanisms exploited by the tumor itself. Knock down of Inhibitor of differentiation protein 2 (Id2-kd) in mouse neuroblastoma whole tumor cells rendered these cells immunogenic. Id2-kd neuroblastoma (Neuro2a) cells (Id2-kd N2a) failed to grow in most immune competent mice and these mice subsequently developed immunity against further wild-type Neuro2a tumor cell challenge. Id2-kd N2a cells grew aggressively in immune-compromised hosts, thereby establishing the immunogenicity of these cells. Therapeutic vaccination with Id2-kd N2a cells alone suppressed tumor growth even in established neuroblastoma tumors and when used in combination with immune checkpoint blockade eradicated large established tumors. Mechanistically, immune cell depletion studies demonstrated that while CD8+ T cells are critical for antitumor immunity, CD4+ T cells are also required to induce a sustained long-lasting helper effect. An increase in number of CD8+ T-cells and enhanced production of interferon gamma (IFNγ) was observed in tumor antigen stimulated splenocytes of vaccinated mice. More importantly, a massive influx of cytotoxic CD8+ T-cells infiltrated the shrinking tumor following combined immunotherapy. These findings show that down regulation of Id2 induced tumor cell immunity and in combination with checkpoint blockade produced a novel, potent, T-cell mediated tumor vaccine strategy.

Abstract A240: A mechanism linking Id2-TGFβ crosstalk to reversible adaptive plasticity in neuroblastoma.

The ability of high-risk neuroblastoma to survive unfavorable growth conditions and multimodal therapy has produced an elusive childhood cancer with a remarkably poor prognosis. A novel phenomenon enabling neuroblastoma to survive selection pressure is its capacity for reversible adaptive plasticity. This plasticity allows cells to transition between highly proliferative anchorage dependent (AD) and slow growing anoikis resistant anchorage independent (AI) phenotypes. The differential gene expression profile of the two cellular phenotypes in the mouse Neuro2a cell line delineated pathways of proliferation in AD cells or tyrosine kinase activation/ apoptosis inhibition in AI cells. A 20 fold overexpression of inhibitor of differentiation 2 (Id2) was identified in AD cells while up-regulation of genes involved in anoikis resistance like PI3K/Akt, Erk, Bcl2, and integrins was observed in AI cells. Similarly, differential expression of Id2 and other genes of interest were also observed in the AD and AI phenotypes of human neuroblastoma cell lines, SK-N-SH and IMR-32; as well as in primary human tumor specimens. Forced down-regulation of Id2 in AD cells or overexpression in AI cells induced the cells to gain characteristics of the other phenotype. Id2 binds both TGFβ and Smad2/3 and appears critical for maintaining the proliferative phenotype at least partially through negative regulation of the TGFβ/ Smad pathway. Simultaneously targeting the differential molecular pathways governing reversible adaptive plasticity resulted in cure of established tumors in a mouse model of neuroblastoma. We present a mechanism that accounts for reversible adaptive plasticity and a molecular basis for combined targeted therapies in neuroblastoma. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A240. Citation Format: Lina Chakrabarti, Bi-Dar Wang, Norman H. Lee, Anthony Sandler. A mechanism linking Id2-TGFβ crosstalk to reversible adaptive plasticity in neuroblastoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A240.