Nicolas Stifani

Motor neurons and the generation of spinal motor neuron diversity

Motor neurons (MNs) are neuronal cells located in the central nervous system (CNS) controlling a variety of downstream targets. This function infers the existence of MN subtypes matching the identity of the targets they innervate. To illustrate the mechanism involved in the generation of cellular diversity and the acquisition of specific identity, this review will focus on spinal MNs (SpMNs) that have been the core of significant work and discoveries during the last decades. SpMNs are responsible for the contraction of effector muscles in the periphery. Humans possess more than 500 different skeletal muscles capable to work in a precise time and space coordination to generate complex movements such as walking or grasping. To ensure such refined coordination, SpMNs must retain the identity of the muscle they innervate. Within the last two decades, scientists around the world have produced considerable efforts to elucidate several critical steps of SpMNs differentiation. During development, SpMNs emerge from dividing progenitor cells located in the medial portion of the ventral neural tube. MN identities are established by patterning cues working in cooperation with intrinsic sets of transcription factors. As the embryo develop, MNs further differentiate in a stepwise manner to form compact anatomical groups termed pools connecting to a unique muscle target. MN pools are not homogeneous and comprise subtypes according to the muscle fibers they innervate. This article aims to provide a global view of MN classification as well as an up-to-date review of the molecular mechanisms involved in the generation of SpMN diversity. Remaining conundrums will be discussed since a complete understanding of those mechanisms constitutes the foundation required for the elaboration of prospective MN regeneration therapies.

Spinal microcircuits comprising dI3 interneurons are necessary for motor functional recovery following spinal cord transection

The spinal cord has the capacity to coordinate motor activities such as locomotion. Following spinal transection, functional activity can be regained, to a degree, following motor training. To identify microcircuits involved in this recovery, we studied a population of mouse spinal interneurons known to receive direct afferent inputs and project to intermediate and ventral regions of the spinal cord. We demonstrate that while dI3 interneurons are not necessary for normal locomotor activity, locomotor circuits rhythmically inhibit them and dI3 interneurons can activate these circuits. Removing dI3 interneurons from spinal microcircuits by eliminating their synaptic transmission left locomotion more or less unchanged, but abolished functional recovery, indicating that dI3 interneurons are a necessary cellular substrate for motor system plasticity following transection. We suggest that dI3 interneurons compare inputs from locomotor circuits with sensory afferent inputs to compute sensory prediction errors that then modify locomotor circuits to effect motor recovery. DOI: http://dx.doi.org/10.7554/eLife.21715.001

O6-Methylguanine-DNA Methyltransferase is a Novel Negative Effector of Invasion in Glioblastoma Multiforme

The dismal prognosis of glioblastoma multiforme (GBM) is mostly due to the high propensity of GBM tumor cells to invade. We reported an inverse relationship between GBM angiogenicity and expression of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT), which has been extensively characterized for its role in resistance to alkylating agents used in GBM treatment. In the present study, given the major role of angiogenesis and invasion in GBM aggressiveness, we aimed to investigate the relationship between MGMT expression and GBM invasion. Stable overexpression of MGMT in the U87MG cell line significantly decreased invasion, altered expression of invasion-related genes, decreased expression of α5β1 integrin and focal adhesion kinase, and reduced their spindle-shaped morphology and migration compared with the empty vector control. Conversely, short hairpin RNA-mediated stable knockdown of MGMT or its pharmacologic depletion in the MGMT-positive T98G cell line were required for increased invasion. The inverse relationship between MGMT and invasion was further validated in primary GBM patient-derived cell lines. Using paraffin-embedded tumors from patients with newly diagnosed GBM (n = 59), tumor MGMT promoter hypermethylation (MGMT gene silencing) was significantly associated with increased immunohistochemical expression of the proinvasive matricellular protein secreted protein acidic and rich in cysteine (SPARC; P = 0.039, χ2 test). Taken together, our findings highlight for the first time the role of MGMT as a negative effector of GBM invasion. Future studies are warranted to elucidate the role of SPARC in the molecular mechanisms underlying the inverse relationship between MGMT and GBM invasion and the potential use of MGMT and SPARC as biomarkers of GBM invasion. Mol Cancer Ther; 11(11); 2440–50. ©2012 AACR.

Kinematic gait parameters are highly sensitive measures of motor deficits and spinal cord injury in mice subjected to experimental autoimmune encephalomyelitis

HighlightsKinematic gait analyses identified movement deficits before the onset of clinical signs in mice subjected to experimental autoimmune encephalomyelitis (EAE).Gait deficits were observed in mice with mild EAE that failed to show clinical disease signs or altered rotarod performance.Impaired movement of the ankle correlated near perfectly with the degree of white matter loss detected in the spinal cord (r = 0.96).Kinematic gait analyses should assist the selection of promising therapeutic candidates for clinical testing. ABSTRACT The preclinical selection of therapeutic candidates for progressive multiple sclerosis (MS) would be aided by the development of sensitive behavioural measures that accurately reflect the impact of autoimmune‐mediated spinal cord damage on locomotion. Neurological deficits in mice subjected to experimental autoimmune encephalomyelitis (EAE) are typically scored using a clinical scale with 5–10 levels of increased disease severity. This ordinal scale represents a general impression of paralysis and impaired gait. By contrast, kinematic gait analyses generate ratio level data that have frequently been used to characterize walking deficits for MS patients and test the efficacy of treatments designed to improve them. Despite these advantages, kinematic gait analyses have not been systematically applied to the study of walking deficits for EAE mice. We have therefore used high speed video recordings (250 frames/s) of EAE mice walking on a treadmill to measure 8 kinematic parameters in the sagittal plane: average hip height (1), average toe height during swing (2), and average angle and range of motion for the hip (3–4), knee (5–6) and ankle (7–8). Kinematic measures of hip, knee and ankle movements were found to be early detectors of impaired locomotion for mice with mild EAE (median clinical score = 1.0 at day post‐immunization 26; DPI 26). These deficits occurred in the absence of reduced rotarod performance with impaired hip and knee movements observed 3 days before disease onset as determined by clinical scores. Gait deficits for mild EAE mice were minor and often recovered fully by DPI 30. By contrast, severe EAE mice (median clinical score = 2.5 at DPI 26) displayed much larger movement impairments for the knee and ankle that failed to completely recover by DPI 44. Moreover, impaired ankle movement was highly correlated with white matter loss in the spinal cords of EAE mice (r = 0.96). Kinematic analyses therefore yield highly sensitive measures of motor deficits that predict spinal cord injury in EAE mice. These behavioural techniques should assist the selection of promising therapeutic candidates for clinical testing in progressive MS.

Genetically identified spinal interneurons integrating tactile afferents for motor control.

Our movements are shaped by our perception of the world as communicated by our senses. Perception of sensory information has been largely attributed to cortical activity. However, a prior level of sensory processing occurs in the spinal cord. Indeed, sensory inputs directly project to many spinal circuits, some of which communicate with motor circuits within the spinal cord. Therefore, the processing of sensory information for the purpose of ensuring proper movements is distributed between spinal and supraspinal circuits. The mechanisms underlying the integration of sensory information for motor control at the level of the spinal cord have yet to be fully described. Recent research has led to the characterization of spinal neuron populations that share common molecular identities. Identification of molecular markers that define specific populations of spinal neurons is a prerequisite to the application of genetic techniques devised to both delineate the function of these spinal neurons and their connectivity. This strategy has been used in the study of spinal neurons that receive tactile inputs from sensory neurons innervating the skin. As a result, the circuits that include these spinal neurons have been revealed to play important roles in specific aspects of motor function. We describe these genetically identified spinal neurons that integrate tactile information and the contribution of these studies to our understanding of how tactile information shapes motor output. Furthermore, we describe future opportunities that these circuits present for shedding light on the neural mechanisms of tactile processing.

Unraveling a locomotor network, many neurons at a time.

In this issue of Neuron, Bruno et al. (2015) use large-scale recordings in Aplysia, and apply novel dimensionality-reduction techniques to define dynamical building blocks involved in locomotor behavior. These techniques open new avenues to the study of neuronal networks.

Robust 3D reconstruction and mean-shift clustering of motoneurons from serial histological images

Motoneurons (MNs) are neuronal cells involved in several central nervous system (CNS) diseases. In order to develop new treatments and therapies, there is a need to understand MN organization and differentiation. Although recently developed embryo mouse models have enabled the investigation of the MN specialization process, more robust and reproducible methods are required to evaluate the topology and structure of the neuron bundles. In this article, we propose a new fully automatic approach to identify MN clusters from stained histological slices. We developed a specific workflow including inter-slice intensity normalization and slice registration for 3D volume reconstruction, which enables the segmentation, mapping and 3D visualization of MN bundles. Such tools will facilitate the understanding of MN organization, differentiation and function.

Abstract 2493: Identification of MGMT-binding proteins involved in the negative regulation of angiogenesis and invasion.

Background: Glioblastoma multiforme (GBM) is the most frequent and most aggressive form of primary malignant brain tumors in adults. The dismal prognosis of GBM patients stems from the highly angiogenic and invasive behavior of GBM tumor cells. O 6 -methylguanine-DNA methyltransferase (MGMT), a DNA repair protein ubiquitously expressed in normal tissues has been extensively characterized for its role in resistance to alkylating agents used in GBM treatment. We reported for the first time an inverse relationship between expression of MGMT and the angiogenic and invasive profile of GBM cell lines. The mechanisms by which MGMT affects angiogenesis and invasion are unknown. We hypothesized that interactions of MGMT with binding proteins (BPs) may account for additional functions beyond its known role as a DNA repair protein. Methods: As a first screening to identify MGMT-BPs with a functional relevance for invasion and angiogenesis, we performed affinity purification of MGMT-BPs following overexpression of FLAG-tagged MGMT and mass spectrometry analysis using 293T-Flag/MGMT and control Flag-tagged empty vector (293T-Flag/EV). Lysates were subjected to affinity purification using an anti-Flag monoclonal antibody covalently attached to agarose resin. The affinity bound FLAG fusion proteins were eluted and separated on SDS-PAGE. Coomassie Blue staining enabled the identification of 6 bands including Flag-MGMT in 293T-Flag/MGMT but not the Flag/EV control. The bands were excised from the gel, subjected to trypsin digestion and identified by liquid chromatography-tandem mass spectrometry. The resultant MS/MS spectra were searched against a proteome database for peptide matching and protein identification. Proteins were identified with high confidence using Scaffold software. Results: Our analysis provided evidence for binding of MGMT to 120 BPs. Using gene ontology (GO) database to search for functional categories, we identified proteins involved in DNA repair, ubiquitin pathway, DNA replication and transcription, RNA metabolism and processing, cell cycle and division, response to stress and cell death. Importantly, we identified proteins involved in cell motility and/or angiogenesis, cytoskeletal-related proteins (15 proteins), small GTPases family and their regulators (10 proteins, such as Rho guanine nucleotide exchange factor 2) and two proteins involved in angiogenesis (Endoribonuclease Dicer and Ribonuclease inhibitor). We also used T98G a human GBM cell line with constitutive expression of MGMT to perform immunoprecipitation of endogenous MGMT (anti-MGMT antibody or the IgG1 isotype control). Mass spectrometry and proteomic analysis of MGMT-BPs in T98G is underway. Conclusion: Our data provide new structural aspects of MGMT and shed light into the multifaceted role of MGMT, which may lead to the identification of novel therapeutic targets in GBM. Citation Format: Siham Sabri, Yaoxian Xu, Nicolas Stifani, Bassam S. Abdulkarim. Identification of MGMT-binding proteins involved in the negative regulation of angiogenesis and invasion. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2493. doi:10.1158/1538-7445.AM2013-2493