University of Vermont

Neuroscience Graduate Program

Matthew Rand

MATTHEW D. RAND

Research Assistant Professor

Anatomy and Neurobiology

Ph.D., Biochemistry, University of Vermont, 1995
Postdoctoral training: Clinical Chemistry, Lund University, Malmo, Sweden; Cell Biology, Harvard Medical School/Cancer Center, Massachussetts General Hospital

Matthew.Rand@uvm.edu
(802) 656-0405

University of Vermont
HSRF 426C
149 Beaumont Ave.
Burlington, VT 05405

Recent research in the news:
Of Fish and Fruit Flies: Recent grant funds research to discover how mercury harms the developing nervous system

 RESEARCH

The overall goal of our research is to identify fundamental mechanisms of neural development that are preferentially sensitive to perturbation by environmental factors. The health and fitness of our nervous system is impingent upon developmental processes that initially generate a vast number of neuronal cells and subsequently maintain the multitude of connections they engage in.  These connections, which are the cellular underpinnings of sensation, movement, behavior and emotion, form in a remarkably precise fashion.  The birth and “wiring up” of neurons requires cell-cell communication that is conveyed by several distinct classes of receptor and ligand molecules, each being at the core of multi-protein signaling cascades.  Given the inherent complexity of how these signals are propagated it is easy to appreciate how even subtle disruption of the earliest stages of development of the nervous system can render debilitating neurological symptoms.

Towards our goal we are currently investigating molecular and cellular mechanisms that orchestrate the normal development and wiring-up of the nervous system and the susceptibility of these mechanisms to environmental insult.  Our studies focus on the Notch signal transduction pathway, a highly conserved mechanism of cell-cell communication.  Once activated by its ligand, Delta, the Notch receptor, engages in signaling in several developmental contexts, most notably in directing neuronal cell fate, influencing neurite morphology and regulating glial cell maturation.  These processes are exquisitely sensitive to the levels of Notch signals.  Recent studies have revealed that proteolysis is a central mechanism in regulating Notch signaling with both Notch and Delta being substrates for ADAM proteases.  ADAMs are cell surface metalloproteases known for their ability to cleave and release the extracellular component of receptor proteinsOur previous work has shown that Kuzbanian (the homolog of mammalian ADAM10) debilitates Delta signaling activity (Qi, et al, Mishra-Gorur, et al). In contrast, proteolysis of Notch by an ADAM is essential for receptor activation.  Thus, ADAMs represent an important molecular partner which can modulate Notch signaling.  In one project in the lab we are investigating the efficacy of each of the five Drosophila ADAM gene products in proteolysis and activity of Notch and Delta to elucidate the overall role that proteolysis plays in regulating this essential pathway.

An integral project in the lab is our ongoing investigation of the developmental effects of the prevalent neurotoxin methylmercury (MeHg).  Inorganic mercury (Hg++) deposited from the atmosphere is converted to MeHg+ by microorganisms of wetlands.  Subsequent bioaccumulation through the food chain results in elevated levels of MeHg in dietary fish in both freshwater and ocean species.  As a result human exposure to MeHg occurs primarily through fish consumption.  The developing fetal nervous system is an exceptionally sensitive target.  Recent studies of fish eating populations reinforce the observation that prenatal MeHg exposure can illicit neurological deficits.  Yet, an estimated 6% of women of child-bearing age have blood mercury levels exceeding the Environmental Protection Agency’s (EPA) reference dose.  Despite the ongoing health risks posed by MeHg the discrete mechanisms that make the developing nervous system most sensitive to MeHg toxicity are not clear.

We recently discovered that MeHg is an effective activator of the Notch receptor (Bland and Rand, 2006).  This occurs through a mechanism that likely engages ADAM activity.  Thus one goal of this project is to characterize the mechanism by which MeHg activates Notch.  We are implementing biochemical and molecular approaches using sulfhydryl-specific alkylating reagents and site directed mutagenesis to determine specific interactions between MeHg and thiol-presenting proteins in the Notch pathway. The consequences of these modifications are being monitored using real-time RT-PCR assays for the transcriptional targets of Notch.  These studies are aimed at elucidating the molecular basis for the cellular disruption seen in a MeHg-exposed nervous system.

We are also identifying genes associated with tolerance, and alternatively, susceptibility to MeHg toxicity.  We are using the power of the Drosophila model to identify wild type strains and to create artificially selected populations that carry heritable MeHg resistance traits.  Through microarray technology we are beginning to determine the changes in mRNA transcripts in the developing nervous system that occur in response to acute and long-term exposure to MeHg.  The overall goal of these efforts is to further elucidate the mechanism of MeHg toxicity and potentially identify conserved molecular pathways involved in MeHg tolerence/susceptibility in higher organisms, including humans.

In an effort to document how these interactions of MeHg and the Notch pathway affect development of the adult nervous system we have been characterizing the development of adult-specific lineages in the Drosophila central nervous system (CNS).  Using clonal marking (MARCM) analyses Jim Truman and coworkers have revealed 24 distinct lineages of central neurons in the thoracic nerve cord of the Drosophila CNS (Development. 2004 131:5167-84).  We have discovered that the Delta ligand is highly expressed in post-mitotic neurons in seven of these lineages, which give rise to the majority of interneurons that process mechano- and chemosensory inputs from the periphery.  Together, with the Truman Lab we have demonstrated a role for Delta in shaping the immature bundles of neurites in these lineages.  Reduced Delta levels in neurons also gives behavioral phenotypes in chemosensory assays.  We are currently investigating how MeHg affects the morphology and function of these Drosophila CNS neurons to correlate phenotypes with those observed previously in the developing human fetal brain.

 SELECTED PUBLICATIONS

Mahapatra CT, Bond J, Rand DM, Rand MD. (2010). Identification of methylmercury tolerance gene candidates in Drosophila. Toxicol Sci. 116(1):225-38. PMID: 20375079

Rand MD. (2010). Drosophotoxicology: The growing potential for Drosophila in neurotoxicology. Neurotoxicol Teratol. 32(1):74-83. PMID: 19559084

Rand MD, Dao JC, Clason TA. (2009). Methylmercury disruption of embryonic neural development in Drosophila. Neurotoxicology 30(5):794-802. PMID: 19409416

Rand MD, Bland CE, Bond J. (2008). Methylmercury activates enhancer-of-split and bearded complex genes independent of the notch receptor. Toxicol Sci. 104(1):163-76.

Cornbrooks C, Bland C, Williams DW, Truman JW, Rand MD. (2006). Delta expression in post-mitotic neurons identifies distinct subsets of adult lineages in Drosophila. J. Neurobiol. 67(1): 23-38.

Bland CE, and Rand MD (2006). Methylmercury induces activation of Notch signaling. Neurotoxicology 27(6):982-91.

Delwig A, Bland C, Beem-Miller M, Kimberly P, Rand MD. Endocytosis-independent mechanisms of Delta ligand proteolysis. (2006).Exp Cell Res. 312:1345-60.

Bland CE, Kimbrerly C, Rand MD (2003). Notch induced proteolysis and nuclear localization of the Delta ligand. J. Biol Chem. 278:13607-10.

Grandbarbe L, Bouissac J, Rand MD, Hrabe de Angelis M, Artavanis-Tsakonas S, Mohier E (2003). Delta-Notch signaling controls the generation of neurons/glia from neural stem cells in a stepwise process. Development 130:1391-402.

Rand MD, Grimm LM, Artavanis-Tsakonas S, Patriub V, Blacklow SC, Sklar J, Aster J (2000). Calcium depletion dissociates and activates heterodimeric Notch receptors. Mol. Cell. Biol. 20:1825-1835.

Artavanis-Tsakonas S, Rand MD, Lake RJ (1999). Notch signaling: cell fate control and signal integration in development. Science 284:770-776.

Qi H*, Rand MD*, Xiaohui W, Wang W, Sestan N, Rakic P, Xu T, Artavanis-Tsakonas S (1999). Processing of the Notch ligand Delta by the metalloprotease Kuzbanian. Science 283:91-94 (*contributed equally).

Rand MD, Lindblom A, Carlson J, Villoutriex B, Stenflo J (1997). Calcium binding to multiple repeats of EGF-like modules. Expression and characterization of the EGF-like modules of human Notch-1 implicated in receptor-ligand interaction. Protein Science 6:2059-2071.

 

The Notch receptor pathway: A model for methylmercury (MeHg)-mediated activation.  Recent studies have documented that Notch activation requires proteolysis that is regeluted by an initial cleavage mediated by an ADAM protease.  Mature Notch is presented at the cell surface as a heterodimeric receptor consisting of the NEC and NTM subunits.  Cleavage by an ADAM protease in the extracellular region of the NTM releases the NEC domain.  Subsequent cleavage in the transmembrane domain, mediated by presenillin g–secretase, yields the Notch intracellular domain (NICD), which translocates to the nucleus and, in complex with the Suppressor of Hairless (Su(H)) co-factor, activates the Enhancer of Split (E(spl)) target gene complex.

ADAM proteases are synthesized as inactive pro-enzymes with an inhibitory propeptide.  A model for Notch activation begins with a high affinity interaction of methylmercury (MeHg) with a conserved cysteine in the ADAM pro-peptide, which unmasks the active site.  The resulting increase in ADAM-mediated proteolysis leads to activation of the Notch receptor.

Methylmercury activates Notch signaling. Using quantitative real-time PCR analyses a dose-dependent increase of the Notch target gene, Enhancer of Split (E(spl)Mg), is seen in a Drosophila neural derived cell line (C3 cells) with the indicated MeHg treatments.  Western blot analyses (right panels) shows the increased proteolytic processing of the Notch receptor in response to MeHg treatment.  An increase in a fragment that co-migrates with NTM subunit is seen with antibody directed to the cytosolic domain, indicative of Notch proteolysis.  In addition, the NEC fragment, detected with an antibody to the extracellular domain, is seen to accumulate in the media of C3 cells with MeHg treatment.
Delta expression in neurite bundles of the thoracic ganglia. A) A schematic representation of the spatial orientation of the parent neuroblasts (NBs) of each Delta-expressing lineage (Delta-lineage NBs in green). B) A diagram illustrating the region of the thoracic ventral nerve cord imaged in C-E. The red arrow represents orientation of view toward the projections in C-E. C-E) A projection of 3-D reconstruction of serial Z-stack images of the T3 neuromere stained for Delta (green, 10D5 antibody) and the pan-neurite marker Neuroglian (purple, BP104 antibody), which identifies the bundles of every lineage. The dorsal one-third of the ganglion is cropped from this image. Neurite bundles of Delta positive lineages 4, 9, 10, 13 and 14 are identified as well as the 22 cluster. Overlap with Neuroglian expression gives the white appearance in panel D. Intense Delta staining is seen in the lateral leg neuropil (LN) where all the Delta lineages (except 10) converge. In several instances the intense green Delta staining overwhelms the Neuroglian staining, particularly at bundle terminal in the leg neuropil. The large Neuroglian-positive peripheral nerves (PN) are seen exiting from the lateral border of the segment. Overall, the limited number of Delta positive lineages relative to the entire ganglion can be appreciated. It is also of note that all the Delta lineages (except 10) project to the ventral neuropil, consistent with a function in sensory processing.
Licorice, the family dog.

Last modified October 10 2011 02:30 PM