Honors & Awards
Basil O'Connor Award, March of Dimes (2003-05)
Brain and Immuno imaging Grant, Dana Foundation (2007-09)
Residency:UCSF School of Medicine (1995) CA
Board Certification: Neurology, American Board of Psychiatry and Neurology (1998)
Internship:UCSF School of Medicine (1992) CA
Medical Education:Emory University Hospital (1991) GA
Residency, UCSF, Neurology (1995)
MD, Emory University, Medicine (1991)
BA, Yale University, Mol Biochem and Biophysics (1985)
Current Research and Scholarly Interests
Reimer Lab interests
A primary interest of our lab is to understand how nerve cells make and recycle neurotransmitters, the small molecules that they use to communicate with each other. In better defining these processes we hope to achieve our long-term goal of identifying novel sites for treatment of diseases such as epilepsy and Parkinson Disease. In our studies on neurotransmitter metabolism we have focused our efforts on transporters, a functional class of proteins that move neurotransmitters and other small molecules across membranes in cells. Transporters have many characteristics that make them excellent pharmacological targets, and not surprisingly some of the most effective treatments for neuropsychiatric disorders are directed at transporters. We are specifically focusing on two groups of transporters vesicular neurotransmitter transporters that package neurotransmitters into vesicles for release, and glutamine transporters that shuttle glutamine, a precursor for two major neurotransmitters glutamate and GABA, to neurons from glia, the supporting cells that surround them. We are pursuing these goals through molecular and biochemical studies, and, in collaboration with the Huguenard and Prince labs, through physiological and biosensor based imaging studies to better understand how pharmacological targeting of these molecules will influence neurological disorders.
A second interest of our lab is to define mechanism underlying the pathology of lysosomal storage disorders. Lysosomes are membrane bound acidic intracellular organelles filled with hydrolytic enzymes that normally function as recycling centers within cells by breaking down damaged cellular macromolecules. Several degenerative diseases designated as lysosomal storage disorders (LSDs) are associated with the accumulation of material within lysosomes. Tay-Sachs disease, Neimann-Pick disease and Gaucher disease are some of the more common LSDs. For reasons that remain incompletely understood, these diseases often affect the nervous system out of proportion to other organs. As a model for LSDs we are studying the lysosomal free sialic acid storage disorders. These diseases are the result of a defect in transport of sialic acid across lysosomal membranes and are associated with mutations in the gene encoding the sialic acid transporter sialin. We are using molecular, genetic and biochemical approaches to better define the normal function of sialin and to determine how loss of sialin function leads to neurodevelopmental defects and neurodegeneration associated with the lysosomal free sialic acid storage disorders.
- Molecular Mechanisms of Neurodegenerative Disease
BIO 267, GENE 267, NENS 267 (Win)
Independent Studies (9)
- Directed Reading in Neurology and Neurological Science
NENS 299 (Aut, Win, Spr)
- Directed Reading in Neurosciences
NEPR 299 (Aut, Win, Spr)
- Early Clinical Experience in Neurology and Neurological Sciences
NENS 280 (Aut, Win, Spr)
- Graduate Research
NENS 399 (Aut, Win, Spr)
- Graduate Research
NEPR 399 (Aut, Win, Spr)
- Medical Scholars Research
NENS 370 (Aut, Win, Spr)
- Out-of-Department Advanced Research Laboratory in Experimental Biology
BIO 199X (Sum)
- Out-of-Department Graduate Research
BIO 300X (Aut, Sum)
- Undergraduate Research
NENS 199 (Aut, Win, Spr)
- Directed Reading in Neurology and Neurological Science
- Prior Year Courses
Biochemistry to the Rescue: A CIC-2 Auxiliary Subunit Provides a Tangible Link to Leukodystrophy
2012; 73 (5): 855-857
ClC-2 is a broadly distributed chloride channel with an enigmatic neurophysiological function. In this issue of Neuron, Jeworutzki et al. (2012) use a biochemical approach to identify GlialCAM, a protein with a defined link to leukodystrophy, as a ClC-2 auxiliary subunit.
View details for DOI 10.1016/j.neuron.2012.02.012
View details for Web of Science ID 000301558600001
View details for PubMedID 22405196
Glutamate biosensor imaging reveals dysregulation of glutamatergic pathways in a model of developmental cortical malformation.
Neurobiology of disease
2012; 49C: 232-246
Cortical malformations can cause intractable epilepsy, but the underlying epileptogenic mechanisms are poorly understood. We used high-speed glutamate biosensor imaging to ask how glutamatergic signaling is altered in cortical malformations induced by neonatal freeze-lesions (FL). In non-lesion neocortical slices from 2 to 8week old rats, evoked glutamate signals were symmetrical in the medio-lateral axis and monotonic, correlating with simple, brief (?50ms) local field potentials (LFPs). By contrast, in FL cortex glutamate signals were prolonged, increased in amplitude, and polyphasic, which paralleled a prolongation of the LFP. Using glutamate biosensor imaging, we found that glutamate signals propagated throughout large areas of FL cortex and were asymmetric (skewed toward the lesion). Laminar analysis demonstrated a shift in the region of maximal glutamate release toward superficial layers in FL cortex. The ability to remove exogenous glutamate was increased within the FL itself but was decreased in immediately adjacent regions. There were corresponding alterations in astrocyte density, with an increase within the lesion and a decrease in deep cortical layers surrounding the lesion. These findings demonstrate both network connectivity and glutamate metabolism are altered in this cortical malformation model and suggests that the regional ability of astrocytes to remove released glutamate may be inversely related to local excitability.
View details for PubMedID 22982711
Structure-Function Studies of the SLC17 Transporter Sialin Identify Crucial Residues and Substrate-induced Conformational Changes
JOURNAL OF BIOLOGICAL CHEMISTRY
2010; 285 (25): 19316-19323
Salla disease and infantile sialic acid storage disorder are human diseases caused by loss of function of sialin, a lysosomal transporter that mediates H(+)-coupled symport of acidic sugars N-acetylneuraminic acid and glucuronic acid out of lysosomes. Along with the closely related vesicular glutamate transporters, sialin belongs to the SLC17 transporter family. Despite their critical role in health and disease, these proteins remain poorly understood both structurally and mechanistically. Here, we use substituted cysteine accessibility screening and radiotracer flux assays to evaluate experimentally a computationally generated three-dimensional structure model of sialin. According to this model, sialin consists of 12 transmembrane helices (TMs) with an overall architecture similar to that of the distantly related glycerol 3-phosphate transporter GlpT. We show that TM4 in sialin lines a large aqueous cavity that forms a part of the substrate permeation pathway and demonstrate substrate-induced alterations in accessibility of substituted cysteine residues in TM4. In addition, we demonstrate that one mutant, F179C, has a dramatically different effect on the apparent affinity and transport rate for N-acetylneuraminic acid and glucuronic acid, suggesting that it may be directly involved in substrate recognition and/or translocation. These findings offer a basis for further defining the transport mechanism of sialin and other SLC17 family members.
View details for DOI 10.1074/jbc.M110.130716
View details for Web of Science ID 000278727800041
View details for PubMedID 20424173
Glutamine Is Required for Persistent Epileptiform Activity in the Disinhibited Neocortical Brain Slice
JOURNAL OF NEUROSCIENCE
2010; 30 (4): 1288-1300
The neurotransmitter glutamate is recycled through an astrocytic-neuronal glutamate-glutamine cycle in which synaptic glutamate is taken up by astrocytes, metabolized to glutamine, and transferred to neurons for conversion back to glutamate and subsequent release. The extent to which neuronal glutamate release is dependent upon this pathway remains unclear. Here we provide electrophysiological and biochemical evidence that in acutely disinhibited rat neocortical slices, robust release of glutamate during sustained epileptiform activity requires that neurons be provided a continuous source of glutamine. We demonstrate that the uptake of glutamine into neurons for synthesis of glutamate destined for synaptic release is not strongly dependent on the system A transporters, but requires another unidentified glutamine transporter or transporters. Finally, we find that the attenuation of network activity through inhibition of neuronal glutamine transport is associated with reduced frequency and amplitude of spontaneous events detected at the single-cell level. These results indicate that availability of glutamine influences neuronal release of glutamate during periods of intense network activity.
View details for DOI 10.1523/JNEUROSCI.0106-09.2010
View details for Web of Science ID 000274050000011
View details for PubMedID 20107056
The Lysosomal Sialic Acid Transporter Sialin Is Required for Normal CNS Myelination
JOURNAL OF NEUROSCIENCE
2009; 29 (49): 15355-15365
Salla disease and infantile sialic acid storage disease are autosomal recessive lysosomal storage disorders caused by mutations in the gene encoding sialin, a membrane protein that transports free sialic acid out of the lysosome after it is cleaved from sialoglycoconjugates undergoing degradation. Accumulation of sialic acid in lysosomes defines these disorders, and the clinical phenotype is characterized by neurodevelopmental defects, including severe CNS hypomyelination. In this study, we used a sialin-deficient mouse to address how loss of sialin leads to the defect in myelination. Behavioral analysis of the sialin(-/-) mouse demonstrates poor coordination, seizures, and premature death. Analysis by histology, electron microscopy, and Western blotting reveals a decrease in myelination of the CNS but normal neuronal cytoarchitecture and normal myelination of the PNS. To investigate potential mechanisms underlying CNS hypomyelination, we studied myelination and oligodendrocyte development in optic nerves. We found reduced numbers of myelinated axons in optic nerves from sialin(-/-) mice, but the myelin that was present appeared grossly normal. Migration and density of oligodendrocyte precursor cells were normal; however, a marked decrease in the number of postmitotic oligodendrocytes and an associated increase in the number of apoptotic cells during the later stages of myelinogenesis were observed. These findings suggest that a defect in maturation of cells in the oligodendrocyte lineage leads to increased apoptosis and underlies the myelination defect associated with sialin loss.
View details for DOI 10.1523/JNEUROSCI.3005-09.2009
View details for Web of Science ID 000272736400003
View details for PubMedID 20007460
Synaptic Vesicle Protein NTT4/XT1 (SLC6A17) Catalyzes Na+-coupled Neutral Amino Acid Transport
JOURNAL OF BIOLOGICAL CHEMISTRY
2009; 284 (13): 8439-8448
The SLC6 family of structurally related, Na(+)-dependent transporter proteins is responsible for presynaptic reuptake of the majority of neurotransmitters. Within this family are a number of orphan transporters, including NTT4/XT1 (SLC6A17), a protein first identified over 15 years ago. NTT4/XT1 is expressed exclusively in the nervous system and specifically on synaptic vesicles in glutamatergic and some GABAergic neurons. Despite extensive efforts by a number of groups, no substrate has been reported for NTT4/XT1. Here we use a combination of molecular manipulations to increase expression of the NTT4/XT1 protein at the plasma membrane and to directly demonstrate that it catalyzes neutral amino acid transport. The substrate profile of the NTT4/XT1-dependent activity is similar to that of the closely related B(0)AT2/SBAT1 (SLC6A15), including a submillimolar apparent affinity for proline and leucine and a low millimolar apparent affinity for glutamine. The transport activity is Na(+)-dependent and Cl(-)-independent and is inhibited by low pH as is SLC6A15, suggesting redundant roles for these proteins. This characterization of NTT4/XT1 offers important insights into neurotransmitter metabolism as well as the mechanistic differences among the structurally related, but functionally divergent, SLC6 proteins.
View details for DOI 10.1074/jbc.M806407200
View details for Web of Science ID 000264397800028
View details for PubMedID 19147495
Imaging of glutamate in brain slices using FRET sensors
JOURNAL OF NEUROSCIENCE METHODS
2008; 168 (2): 306-319
The neurotransmitter glutamate is the mediator of excitatory neurotransmission in the brain. Release of this signaling molecule is carefully controlled by multiple mechanisms, yet the methods available to measure released glutamate have been limited in spatial and/or temporal domains. We have developed a novel technique to visualize glutamate release in brain slices using three purified fluorescence (Forster) energy resonance transfer (FRET)-based glutamate sensor proteins. Using a simple loading protocol, the FRET sensor proteins diffuse deeply into the extracellular space and remain functional for many tens of minutes. This allows imaging of glutamate release in brain slices with simultaneous electrophysiological recordings and provides temporal and spatial resolution not previously possible. Using this glutamate FRET sensor loading and imaging protocol, we show that changes in network excitability and glutamate re-uptake alter evoked glutamate transients and produce correlated changes in evoked-cortical field potentials. Given the sophisticated advantages of brain slices for electrophysiological and imaging protocols, the ability to perform real-time imaging of glutamate in slices should lead to key insights in brain function relevant to plasticity, development and pathology. This technique also provides a unique assay of network activity that compliments alternative techniques such as voltage-sensitive dyes and multi-electrode arrays.
View details for DOI 10.1016/j.jneumeth.2007.10.017
View details for Web of Science ID 000253824400004
View details for PubMedID 18160134
G328E and G409E sialin missense mutations similarly impair transport activity, but differentially affect trafficking
MOLECULAR GENETICS AND METABOLISM
2007; 92 (4): 371-374
Two disease-associated missense mutations in the sialin gene (G328E and G409E) have recently been identified in patients with lysosomal free sialic acid storage disease. We have assessed the effect of these mutations and find complete loss of measurable transport activity with both and impaired trafficking of the G409E protein. These results suggest that the two residues are important for proper function of sialin and confirm the association of loss of transport with disease causative mutations.
View details for DOI 10.1016/j.ymgme.2007.08.121
View details for Web of Science ID 000252054000012
View details for PubMedID 17933575
Modulation of epileptiform activity by glutamine and system A transport in a model of post-traumatic epilepsy
NEUROBIOLOGY OF DISEASE
2007; 25 (2): 230-238
Epileptic activity arises from an imbalance in excitatory and inhibitory synaptic transmission. To determine if alterations in the metabolism of glutamate, the primary excitatory neurotransmitter, might contribute to epilepsy we directly and indirectly modified levels of glutamine, an immediate precursor of synaptically released glutamate, in the rat neocortical undercut model of hyperexcitability and epilepsy. We show that slices from injured cortex take up glutamine more readily than control slices, and an increased expression of the system A transporters SNAT1 and SNAT2 likely underlies this difference. We also examined the effect of exogenous glutamine on evoked and spontaneous activity and found that addition of physiological concentrations of glutamine to perfusate of slices isolated from injured cortex increased the incidence and decreased the refractory period of epileptiform potentials. By contrast, exogenous glutamine increased the amplitude of evoked potentials in normal cortex, but did not induce epileptiform potentials. Addition of physiological concentrations of glutamine to perfusate of slices isolated from injured cortex greatly increased abnormal spontaneous activity in the form of events resembling spreading depression, again while having no effect on slices from normal cortex. Interestingly, similar spreading depression like events were noted in control slices at supraphysiological levels of glutamine. In the undercut cortex addition of methylaminoisobutyric acid (MeAIB), an inhibitor of the system A glutamine transporters attenuated all physiological effects of added glutamine suggesting that uptake through these transporters is required for the effect of glutamine. Our findings support a role for glutamine transport through SNAT1 and/or SNAT2 in the maintenance of abnormal activity in this in vitro model of epileptogenesis and suggest that system A transport and glutamine metabolism are potential targets for pharmacological intervention in seizures and epilepsy.
View details for DOI 10.1016/j.nbd.2006.08.025
View details for Web of Science ID 000243981400002
View details for PubMedID 17070687
Biochemical and genetic analysis of ANK in arthritis and bone disease
AMERICAN JOURNAL OF HUMAN GENETICS
2006; 79 (6): 1017-1029
Mutations in the progressive ankylosis gene (Ank/ANKH) cause surprisingly different skeletal phenotypes in mice and humans. In mice, recessive loss-of-function mutations cause arthritis, ectopic crystal formation, and joint fusion throughout the body. In humans, some dominant mutations cause chondrocalcinosis, an adult-onset disease characterized by the deposition of ectopic joint crystals. Other dominant mutations cause craniometaphyseal dysplasia, a childhood disease characterized by sclerosis of the skull and abnormal modeling of the long bones, with little or no joint pathology. Ank encodes a multiple-pass transmembrane protein that regulates pyrophosphate levels inside and outside tissue culture cells in vitro, but its mechanism of action is not yet clear, and conflicting models have been proposed to explain the effects of the human mutations. Here, we test wild-type and mutant forms of ANK for radiolabeled pyrophosphate-transport activity in frog oocytes. We also reconstruct two human mutations in a bacterial artificial chromosome and test them in transgenic mice for rescue of the Ank null phenotype and for induction of new skeletal phenotypes. Wild-type ANK stimulates saturable transport of pyrophosphate ions across the plasma membrane, with half maximal rates attained at physiological levels of pyrophosphate. Chondrocalcinosis mutations retain apparently wild-type transport activity and can rescue the joint-fusion phenotype of Ank null mice. Craniometaphyseal dysplasia mutations do not transport pyrophosphate and cannot rescue the defects of Ank null mice. Furthermore, microcomputed tomography revealed previously unappreciated phenotypes in Ank null mice that are reminiscent of craniometaphyseal dysplasia. The combination of biochemical and genetic analyses presented here provides insight into how mutations in ANKH cause human skeletal disease.
View details for Web of Science ID 000242131600003
View details for PubMedID 17186460
Detection of glutamate release from neurons by genetically encoded surface-displayed FRET nanosensors
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2005; 102 (24): 8740-8745
Glutamate is the predominant excitatory neurotransmitter in the mammalian brain. Once released, its rapid removal from the synaptic cleft is critical for preventing excitotoxicity and spillover to neighboring synapses. Despite consensus on the role of glutamate in normal and disease physiology, technical issues limit our understanding of its metabolism in intact cells. To monitor glutamate levels inside and at the surface of living cells, genetically encoded nanosensors were developed. The fluorescent indicator protein for glutamate (FLIPE) consists of the glutamate/aspartate binding protein ybeJ from Escherichia coli fused to two variants of the green fluorescent protein. Three sensors with lower affinities for glutamate were created by mutation of residues peristeric to the ybeJ binding pocket. In the presence of ligands, FLIPEs show a concentration-dependent decrease in FRET efficiency. When expressed on the surface of rat hippocampal neurons or PC12 cells, the sensors respond to extracellular glutamate with a reversible concentration-dependent decrease in FRET efficiency. Depolarization of neurons leads to a reduction in FRET efficiency corresponding to 300 nM glutamate at the cell surface. No change in FRET was observed when cells expressing sensors in the cytosol were superfused with up to 20 mM glutamate, consistent with a minimal contribution of glutamate uptake to cytosolic glutamate levels. The results demonstrate that FLIPE sensors can be used for real-time monitoring of glutamate metabolism in living cells, in tissues, or in intact organisms, providing tools for studying metabolism or for drug discovery.
View details for Web of Science ID 000229807200061
View details for PubMedID 15939876
Varied mechanisms underlie the free sialic acid storage disorders
JOURNAL OF BIOLOGICAL CHEMISTRY
2005; 280 (2): 1408-1416
Salla disease and infantile sialic acid storage disorder are autosomal recessive neurodegenerative diseases characterized by loss of a lysosomal sialic acid transport activity and the resultant accumulation of free sialic acid in lysosomes. Genetic analysis of these diseases has identified several unique mutations in a single gene encoding a protein designated sialin (Verheijen, F. W., Verbeek, E., Aula, N., Beerens, C. E., Havelaar, A. C., Joosse, M., Peltonen, L., Aula, P., Galjaard, H., van der Spek, P. J., and Mancini, G. M. (1999) Nat. Genet. 23, 462-465; Aula, N., Salomaki, P., Timonen, R., Verheijen, F., Mancini, G., Mansson, J. E., Aula, P., and Peltonen, L. (2000) Am. J. Hum. Genet. 67, 832-840). From the biochemical phenotype of the diseases and the predicted polytopic structure of the protein, it has been suggested that sialin functions as a lysosomal sialic acid transporter. Here we directly demonstrate that this activity is mediated by sialin and that the recombinant protein has functional characteristics similar to the native lysosomal sialic acid transport system. Furthermore, we describe the effect of disease-causing mutations on the protein. We find that the majority of the mutations are associated with a complete loss of activity, while the mutations associated with the milder forms of the disease lead to reduced, but residual, function. Thus, there is a direct correlation between sialin function and the disease state. In addition, we find with one mutation that the protein is retained in the endoplasmic reticulum, indicating that altered trafficking of sialin is also associated with disease. This analysis of the molecular mechanism of sialic acid storage disorders is a further step in identifying therapeutic approaches to these diseases.
View details for DOI 10.1074/jbc.M411295200
View details for Web of Science ID 000226195200068
View details for PubMedID 15516337
Organic anion transport is the primary function of the SLC17/type I phosphate transporter family
PFLUGERS ARCHIV-EUROPEAN JOURNAL OF PHYSIOLOGY
2004; 447 (5): 629-635
Recently, molecular studies have determined that the SLC17/type I phosphate transporters, a family of proteins initially characterized as phosphate carriers, mediate the transport of organic anions. While their role in phosphate transport remains uncertain, it is now clear that the transport of organic anions facilitated by this family of proteins is involved in diverse processes ranging from the vesicular storage of the neurotransmitter glutamate to the degradation and metabolism of glycoproteins.
View details for DOI 10.1007/s00424-003-1087-y
View details for Web of Science ID 000188837300018
View details for PubMedID 12811560