Integrative analysis identifies candidate tumor microenvironment and intracellular signaling pathways that define tumor heterogeneity in NF1

Neurofibromatosis type 1 (NF1) is a monogenic syndrome that gives rise to numerous symptoms including cognitive impairment, skeletal abnormalities, and growth of benign nerve sheath tumors. Nearly all NF1 patients develop cutaneous neurofibromas (cNFs), which occur on the skin surface, whereas 40-60% of patients develop plexiform neurofibromas (pNFs), which are deeply embedded in the peripheral nerves. Patients with pNFs have a ~10% lifetime chance of these tumors becoming malignant peripheral nerve sheath tumors (MPNSTs). These tumors have a severe prognosis and few treatment options other than surgery. Given the lack of therapeutic options available to patients with these tumors, identification of druggable pathways or other key molecular features could aid ongoing therapeutic discovery studies. In this work, we used statistical and machine learning methods to analyze 77 NF1 tumors with genomic data to characterize key signaling pathways that distinguish these tumors and identify candidates for drug development. We identified subsets of latent gene expression variables that may be important in the identification and etiology of cNFs, pNFs, other neurofibromas, and MPNSTs. Furthermore, we characterized the association between these latent variables and genetic variants, immune deconvolution predictions, and protein activity predictions

A Study of the Different Phases of Refinement of an Inhibitory Circuit in the Mouse Auditory Brainstem

<p>Crudely assembled neuronal circuits with exuberant innervations are refined into precise adult-like circuits by functional silencing and structural pruning of the surplus connections during early development. Such reorganizations are evident in several excitatory circuits as well as in the inhibitory circuit between the medial nucleus of trapezoid body (MNTB) and the lateral superior olive (LSO). LSO neurons integrate excitation from the glutamatergic inputs from ipsilateral anteroventral cochlear nucleus (AVCN) and inhibition from the contralateral AVCN via GABA/glycinergic inputs from the ipsilateral MNTB (Cant and Casseday, 1986; Spangler et al., 1985). The tonotopic arrangement of the circuit ensures that the excitatory and inhibitory inputs corresponding to the same sound frequency converge on to the same population of LSO neurons. Due to this tonotopic arrangement, the medial part of the circuit responds to high frequency sound stimulus and the lateral part of the circuit responds to low frequency sound stimulus (Kandler and Friauf, 1993; Friauf, 1992; Tsuchitani, 1977). In the first two postnatal weeks of pre-hearing development, the medial part of the circuit undergoes extensive functional refinement during which single LSO neurons lose 75 % of their initial MNTB connections and the remaining inputs are strengthened by 8 fold (Kim and Kandler, 2003; Noh et al., 2010). The neurotransmitter phenotype of this circuit also transforms from a GABA/Glycine/Glutamate co-release to primarily glycinergic release during this period (Kotak et al., 1998; Gillespie et al., 2005). However, it is still unclear whether functional elimination and strengthening of inputs occurs simultaneously or in succession and which factors influence these processes. In this study I investigated the course of refinement of the medial and lateral MNTB inputs to the LSO in acute brainstem slices of E 18 to P 13 mice using whole-cell patch clamp technique. I determined the strength of single MNTB inputs using minimal stimulation technique. I estimated the number of inputs on to each LSO cell by calculating the convergence ratio derived from the postsynaptic response amplitudes to minimal and maximal stimulation of MNTB fibers. I also investigated the role of transient glutamatergic and GABAergic co-release from the MNTB inputs in the process of refinement and strengthening of the inputs. I observed three distinct phases in the pre-hearing refinement of the MNTB-LSO circuit. The first phase or the proliferation phase (between embryonic day 18 and postnatal day (P) 2-3) is followed by a functional elimination phase (between P 3 and P5). This is then followed by a strengthening phase (betweenP6 and P9) of the retained MNTB inputs. I observed extensive proliferation and functional elimination of inputs in the medial part of the circuit while no significant elimination occurred in the lateral part. Both medial and lateral inputs strengthened about 3-4 fold in the first two weeks of development. I also observed that the transient glutamate co-release, which is essential for the refinement and strengthening of the medial MNTB-2 LSO projections, does not seem to play an important role in the strengthening of the lateral MNTB inputs. I further investigated the role of GABA co-release in the process of refinement of the MNTB-LSO circuit using a conditional Gad1 knockout mouse. However, the study of the role of GABA co-release in the MNTB-LSO circuit was inconclusive due to the lack of phenotypic alteration of the GABAergic input in the MNTB-LSO circuit of the Gad1 knockout mice. Thus from the present study it is clear that, during the pre-hearing development, functional elimination of specific inputs occurs before strengthening of the inputs in the medial MNTB-LSO circuit. So the strength of single inputs does not seem to be a key factor in determining which inputs are functionally eliminated. Significant strengthening occurs in the lateral MNTB-LSO circuit even in the absence of any functional elimination. Further, while glutamate co-release in the circuit seems to be crucial for the elimination and strengthening of medial MNTB-LSO projections, it does not seem to be important for the lateral part of the circuit. These observations collectively suggest that the functional elimination phase and the strengthening phase in the pre-hearing refinement of the MNTB-LSO circuit seem to be independent of each other with different underlying mechanisms.</p

Immediate Effects of Repetitive Magnetic Stimulation on Single Cortical Pyramidal Neurons.

Repetitive Transcranial Magnetic Stimulation (rTMS) has been successfully used as a non-invasive therapeutic intervention for several neurological disorders in the clinic as well as an investigative tool for basic neuroscience. rTMS has been shown to induce long-term changes in neuronal circuits in vivo. Such long-term effects of rTMS have been investigated using behavioral, imaging, electrophysiological, and molecular approaches, but there is limited understanding of the immediate effects of TMS on neurons. We investigated the immediate effects of high frequency (20 Hz) rTMS on the activity of cortical neurons in an effort to understand the underlying cellular mechanisms activated by rTMS. We used whole-cell patch-clamp recordings in acute rat brain slices and calcium imaging of cultured primary neurons to examine changes in neuronal activity and intracellular calcium respectively. Our results indicate that each TMS pulse caused an immediate and transient activation of voltage gated sodium channels (9.6 ± 1.8 nA at -45 mV, p value < 0.01) in neurons. Short 500 ms 20 Hz rTMS stimulation induced action potentials in a subpopulation of neurons, and significantly increased the steady state current of the neurons at near threshold voltages (at -45 mV: before TMS: I = 130 ± 17 pA, during TMS: I = 215 ± 23 pA, p value = 0.001). rTMS stimulation also led to a delayed increase in intracellular calcium (153.88 ± 61.94% increase from baseline). These results show that rTMS has an immediate and cumulative effect on neuronal activity and intracellular calcium levels, and suggest that rTMS may enhance neuronal responses when combined with an additional motor, sensory or cognitive stimulus. Thus, these results could be translated to optimize rTMS protocols for clinical as well as basic science applications

rTMS increases steady state current in cortical neurons.

<p>(A) Example voltage clamp traces of (top) stimulus and (bottom) responses (before and during TMS) in cortical neurons (traces have been filtered to remove the TMS artifact). The red dotted line in the response traces denotes the point at which steady state current was measured. (B) Population data of steady state current of cortical neurons at different voltages (Numbers in the bars indicate sample size). Error bars represent SEM.</p

rTMS induces action potentials and reshapes spike timing.

<p>(A) Example trace of a neuron showing lower activation threshold during rTMS stimulation (i) 50 pA subthreshold stimulus step (ii) response before rTMS, (iii) response during rTMS (B) Example trace of a neuron showing the effect of rTMS on spike timing (i) 300 pA supratheshold stimulus step (ii) response before rTMS, (iii) response during rTMS. (C) Upper panel: Magnified view of an action potential before TMS (corresponding to grey box in B (ii)), Lower panel: Magnified view of a TMS artifact (arrow head, two pronged artifact) followed by an action potential during stimulation (corresponding to grey box in B (iii)), (D) Representative post stimulus time histogram (PSTH) of the response neuron in (B) during rTMS stimulation.</p

TMS pulses transiently open voltage gated sodium channels.

<p>(A) Example voltage clamp traces of TMS induced responses in cortical cells at -45mV in the presence and absence of DNQX, DL-AP5, and SR99531 which are pharmacological agents blocking glutamatergic and GABAergic signaling in the slice. (B) Population data of the mean amplitude of TMS pulse-induced current in cortical neurons during different pharmacological conditions (Numbers within bars indicate sample size for each experimental group). Error bars represent SEM.</p

rTMS increases intracellular calcium concentrations in cultured neurons.

<p>(A) Example time course plot of the increase in calcium signal in a cultured cortical neuron due to TMS. Grey bar indicates 10 s TMS stimulation. (B) Plot showing peaks of intracellular calcium fluxes (black dots) in individual neurons (n = 23) as they occur after TMS stimulation (grey bar).</p

TMS pulses induce transient inward currents at specific voltages.

<p>(A) Example voltage clamp traces of TMS induced responses in Model Cell (top panel, no neuron) and cortical neurons (bottom panel, TMS + neuron). (B) Average amplitudes of the TMS-induced current in cortical neurons (n = 10) at different voltages. Error bars represent SEM.</p

CX3CL1 Action on Microglia Protects from Diet-Induced Obesity by Restoring POMC Neuronal Excitability and Melanocortin System Activity Impaired by High-Fat Diet Feeding

Both hypothalamic microglial inflammation and melanocortin pathway dysfunction contribute to diet-induced obesity (DIO) pathogenesis. Previous studies involving models of altered microglial signaling demonstrate altered DIO susceptibility with corresponding POMC neuron cytological changes, suggesting a link between microglia and the melanocortin system. We addressed this hypothesis using the specific microglial silencing molecule, CX3CL1 (fractalkine), to determine whether reducing hypothalamic microglial activation can restore POMC/melanocortin signaling to protect against DIO. We performed metabolic analyses in high fat diet (HFD)-fed mice with targeted viral overexpression of CX3CL1 in the hypothalamus. Electrophysiologic recording in hypothalamic slices from POMC-MAPT-GFP mice was used to determine the effects of HFD feeding and microglial silencing via minocycline or CX3CL1 on GFP-labeled POMC neurons. Finally, mice with hypothalamic overexpression of CX3CL1 received central treatment with the melanocortin receptor antagonist SHU9119 to determine whether melanocortin signaling is required for the metabolic benefits of CX3CL1. Hypothalamic overexpression of CX3CL1 increased leptin sensitivity and POMC gene expression, while reducing weight gain in animals fed an HFD. In electrophysiological recordings from hypothalamic slice preparations, HFD feeding was associated with reduced POMC neuron excitability and increased amplitude of inhibitory postsynaptic currents. Microglial silencing using minocycline or CX3CL1 treatment reversed these HFD-induced changes in POMC neuron electrophysiologic properties. Correspondingly, blockade of melanocortin receptor signaling in vivo prevented both the acute and chronic reduction in food intake and body weight mediated by CX3CL1. Our results show that suppressing microglial activation during HFD feeding reduces DIO susceptibility via a mechanism involving increased POMC neuron excitability and melanocortin signaling

Integrative Analysis Identifies Candidate Tumor Microenvironment and Intracellular Signaling Pathways that Define Tumor Heterogeneity in NF1

Neurofibromatosis type 1 (NF1) is a monogenic syndrome that gives rise to numerous symptoms including cognitive impairment, skeletal abnormalities, and growth of benign nerve sheath tumors. Nearly all NF1 patients develop cutaneous neurofibromas (cNFs), which occur on the skin surface, whereas 40&ndash;60% of patients develop plexiform neurofibromas (pNFs), which are deeply embedded in the peripheral nerves. Patients with pNFs have a ~10% lifetime chance of these tumors becoming malignant peripheral nerve sheath tumors (MPNSTs). These tumors have a severe prognosis and few treatment options other than surgery. Given the lack of therapeutic options available to patients with these tumors, identification of druggable pathways or other key molecular features could aid ongoing therapeutic discovery studies. In this work, we used statistical and machine learning methods to analyze 77 NF1 tumors with genomic data to characterize key signaling pathways that distinguish these tumors and identify candidates for drug development. We identified subsets of latent gene expression variables that may be important in the identification and etiology of cNFs, pNFs, other neurofibromas, and MPNSTs. Furthermore, we characterized the association between these latent variables and genetic variants, immune deconvolution predictions, and protein activity predictions