Dr. Reimer specializes in treatment of lysosomal storage disorders that affect the nervous system. He has been practicing as a neurologist for over 20 years. He has a particular interest in Fabry disease and Gaucher disease.

Clinical Focus

  • Neurology

Academic Appointments

Honors & Awards

  • Basil O'Connor Award, March of Dimes (2003-05)
  • Brain and Immuno imaging Grant, Dana Foundation (2007-09)

Professional Education

  • Board Certification: American Board of Psychiatry and Neurology, Neurology (1998)
  • Residency: University of California at San Francisco School of Medicine (1995) CA
  • Internship: University of California at San Francisco School of Medicine (1992) CA
  • MD, Emory University, Medicine (1991)
  • BA, Yale University, Molecular Biophysics and Biochemistry (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.

Clinical Trials

  • The Effect of Exercise on Muscle Dysfunction in Cystinosis Recruiting

    Classification of activity tolerance is of importance in chronic progressive myopathies, not only to better understand functional implications of the disease state itself, but also for purposes of exercise prescription for health maintenance. Maximal exercise testing has been considered as the gold standard of assessing maximal aerobic capacity, however testing in individuals with neuromuscular disease is often limited due to pain, activity intolerance, musculoskeletal impairments, fatigue and other such related variables. Often, submaximal exercise testing can overcome some of these obstacles, and as such, is used frequently in the clinical environment. Non-ambulatory exercise testing utilizing an arm ergometer specifically has not been studied as heavily, especially in those with progressive myopathies. For this study, we will use maximal aerobic capacity testing for individuals with Cystinosis Myopathy utilizing a bike ergometer to allow testing of individuals regardless of their ambulatory status.

    View full details

2023-24 Courses

Stanford Advisees

All Publications

  • Collaborative research efforts drive therapeutic advancements for free sialic acid storage disorder (FSASD) Malicdan, M., Adams, D. R., Brooks, P. J., Dobrenis, K., Gahl, W. A., Gasnier, B., Hackbarth, M. E., Hossain, M., Leoyklang, P., Anne-Longin, C., Paavola, L., Platt, F., Pollard, L., Reimer, R., Sabir, M. S., Wang, R. Y., Walkley, S., Wasserstein, M., Huizing, M. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2024
  • An electronic health record cohort of Veterans with amyotrophic lateral sclerosis. Amyotrophic lateral sclerosis & frontotemporal degeneration Reimer, R. J., Goncalves, A., Soper, B., Cadena, J., Wilson, J. L., Gryshuk, A. L., Suarez, P., Osborne, T. F., Grimes, K. V., Ray, P. 2023: 1-7


    Objective: To assemble and characterize an electronic health record (EHR) dataset for a large cohort of US military Veterans diagnosed with ALS (Amyotrophic Lateral Sclerosis). Methods: An EHR dataset for 19,662 Veterans diagnosed with ALS between January 1, 2000 to December 31, 2020 was compiled from the Veterans Health Administration (VHA) EHR database by a query for ICD9 diagnosis (335.20) or ICD10 diagnosis (G12.21) for Amyotrophic Lateral Sclerosis. Results: The cohort is predominantly male (98.94%) and white (72.37%) with a median age at disease onset of 68 years and median survival from the date of diagnosis of 590 days. With the designation of ALS as a compensable illness in 2009, there was a subsequent increase in the number of Veterans diagnosed per year in the VHA, but no change in median survival. The cohort included a greater-than-expected proportion of individuals whose branch of service at the time of separation was the Army. Conclusions: The composition of the cohort reflects the VHA population who are at greatest risk for ALS. The greater than expected proportion of individuals whose branch of service at the time of separation was the Army suggests the possibility of a branch-specific risk factor for ALS.

    View details for DOI 10.1080/21678421.2023.2239300

    View details for PubMedID 37555559

  • A concerted action to explore therapies for free sialic acid storage disease Huizing, M., Walkley, S. U., Wasserstein, M., Anne-Longin, C., Wang, R. Y., Reimer, R., Pollard, L., Paavola, L., Malicdan, M., Sabir, M. S., Leoyklang, P., Adams, D. R., Gahl, W. A. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2023: 64-65
  • Glial control of sphingolipid levels sculpts diurnal remodeling in a circadian circuit. Neuron Vaughen, J. P., Theisen, E., Rivas-Serna, I. M., Berger, A. B., Kalakuntla, P., Anreiter, I., Mazurak, V. C., Rodriguez, T. P., Mast, J. D., Hartl, T., Perlstein, E. O., Reimer, R. J., Clandinin, M. T., Clandinin, T. R. 2022


    Structural plasticity in the brain often necessitates dramatic remodeling of neuronal processes, with attendant reorganization of the cytoskeleton and membranes. Although cytoskeletal restructuring has been studied extensively, how lipids might orchestrate structural plasticity remains unclear. We show that specific glial cells in Drosophila produce glucocerebrosidase (GBA) to locally catabolize sphingolipids. Sphingolipid accumulation drives lysosomal dysfunction, causing gba1b mutants to harbor protein aggregates that cycle across circadian time and are regulated by neural activity, the circadian clock, and sleep. Although the vast majority of membrane lipids are stable across the day, a specific subset that is highly enriched in sphingolipids cycles daily in a gba1b-dependent fashion. Remarkably, both sphingolipid biosynthesis and degradation are required for the diurnal remodeling of circadian clock neurites, which grow and shrink across the day. Thus, dynamic sphingolipid regulation by glia enables diurnal circuit remodeling and proper circadian behavior.

    View details for DOI 10.1016/j.neuron.2022.07.016

    View details for PubMedID 35961319

  • Metalworking Fluid Exposure and Stroke Mortality Among U.S. Autoworkers. American journal of epidemiology Elser, H., Chen, K. T., Arteaga, D., Reimer, R., Picciotto, S., Costello, S., Eisen, E. A. 1800


    Although Air pollution is an important risk factor for stroke, few studies have considered the impact of workplace exposure to particulate matter (PM). We examined implications of exposure to PM composed of metalworking fluids (MWF) for stroke mortality in the United Autoworkers-General Motors cohort. Cox proportional hazards models with age as the timescale were used to estimate the association of cumulative straight, soluble, and synthetic MWF exposure with stroke mortality controlling for sex, race, plant, calendar year, and hire year. Among 38,553 autoworkers followed 1941-1995, we identified 114 ischemic stroke deaths and 113 hemorrhagic stroke deaths. Overall stroke mortality risk was increased among workers in the middle exposure category for straight MWF (HR: 1.31, 95% CI: 0.87-1.98), and workers in the highest exposure category for synthetic MWF (1.94, 1.13-3.16) compared to workers with no direct exposure. Ischemic stroke mortality risk was increased among workers in the highest exposure categories for straight MWF (1.45, 0.83-2.53) and synthetic MWF (2.50, 1.39-4.50). We observed no clear relationship between MWF exposure and hemorrhagic stroke mortality. Our results support a potentially important role for occupational PM exposures in stroke mortality and indicate the need for further studies of PM exposure and stroke in varied occupational settings.

    View details for DOI 10.1093/aje/kwac002

    View details for PubMedID 35029630

  • Acute on Chronic Neuromuscular Respiratory Failure in the Intensive Care Unit: Optimization of Triage, Ventilation Modes, and Extubation CUREUS Murray, N. M., Reimer, R. J., Cao, M. 2021; 13 (7)
  • Acute on Chronic Neuromuscular Respiratory Failure in the Intensive Care Unit: Optimization of Triage, Ventilation Modes, and Extubation. Cureus Murray, N. M., Reimer, R. J., Cao, M. 2021; 13 (7): e16297


    Critical care management of acute respiratory failure in patients with neuromuscular disease (NMD) such as amyotrophic lateral sclerosis (ALS) is not standardized and is challenging for many critical care specialists. Progressive hypercapnic respiratory failure and ineffective airway clearance are key issues in this patient population. Often at the time of hospital presentation, patients are already supported by home mechanical ventilatory support with noninvasive ventilation (NIV) and an airway clearance regimen. Prognosis is poor once a patient develops acute respiratory failure requiring intubation and invasive mechanical ventilatory support, commonly leading to tracheostomy or palliative-focused care.  We focus on this understudied group of patients with ALS without tracheostomy and incorporate existing data to propose a technical approach to the triage and management of acute respiratory failure, primarily for those who require intubation and mechanical ventilatory support for reversible causes, and also for progression of end-stage disease. Optimizing management in this setting improves both quality and quantity of life. Neuromuscular patients with acute respiratory failure require protocolized and personalized triage and treatment. Here, we describe the technical methods used at our single institution. The triage phase incorporates comprehensive evaluation for new etiologies of hypoxia and hypercapnia, which are not initially presumed to be secondary to progression or end-stage neuromuscular respiratory failure. In select patients, this may involve intubation or advanced adjustments of NIV machines. Next, once the acute etiology(s) is identified and treated, the focus shifts: training and use of mechanical airway clearance to optimize pulmonary function, facilitation of NIV wean or successful extubation to NIV, and transition to a stable regimen for home ventilation. The comprehensive protocol described here incorporates multi-institutional approaches and effectively optimizes acute respiratory failure in patients with neuromuscular pulmonary disease.

    View details for DOI 10.7759/cureus.16297

    View details for PubMedID 34381654

    View details for PubMedCentralID PMC8351614

  • Leigh Disease-Like Neuropathology in an Adult Following Covid-19 Infection and Global Anoxia Sobel, R., Dulken, B., Reimer, R., Louie, C. OXFORD UNIV PRESS INC. 2021: 582
  • Correction to: Air pollution, methane super-emitters, and oil and gas wells in Northern California: the relationship with migraine headache prevalence and exacerbation. Environmental health : a global access science source Elser, H., Morello-Frosch, R., Jacobson, A., Pressman, A., Kioumourtzoglou, M., Reimer, R., Casey, J. A. 2021; 20 (1): 57

    View details for DOI 10.1186/s12940-021-00745-8

    View details for PubMedID 33971885

  • Air pollution, methane super-emitters, and oil and gas wells in Northern California: the relationship with migraine headache prevalence and exacerbation. Environmental health : a global access science source Elser, H., Morello-Frosch, R., Jacobson, A., Pressman, A., Kioumourtzoglou, M., Reimer, R., Casey, J. A. 2021; 20 (1): 45


    BACKGROUND: Migraine-an episodic disorder characterized by severe headache that can lead to disability-affects over 1 billion people worldwide. Prior studies have found that short-term exposure to fine particulate matter (PM2.5), nitrogen dioxide (NO2), and ozone increases risk of migraine-related emergency department (ED) visits. Our objective was to characterize the association between long-term exposure to sources of harmful emissions and common air pollutants with both migraine headache and, among patients with migraine, headache severity.METHODS: From the Sutter Health electronic health record database, we identified 89,575 prevalent migraine cases between 2014 and 2018 using a migraine probability algorithm (MPA) score and 270,564 frequency-matched controls. Sutter Health delivers care to 3.5 million patients annually in Northern California. Exposures included 2015 annual average block group-level PM2.5 and NO2 concentrations, inverse-distance weighted (IDW) methane emissions from 60 super-emitters located within 10km of participant residence between 2016 and 2018, and IDW active oil and gas wells in 2015 within 10km of each participant. We used logistic and negative binomial mixed models to evaluate the association between environmental exposures and (1) migraine case status; and (2) migraine severity (i.e., MPA score>100, triptan prescriptions, neurology visits, urgent care migraine visits, and ED migraine visits per person-year). Models controlled for age, sex, race/ethnicity, Medicaid use, primary care visits, and block group-level population density and poverty.RESULTS: In adjusted analyses, for each 5ppb increase in NO2, we observed 2% increased odds of migraine case status (95% CI: 1.00, 1.05) and for each 100,000kg/hour increase in IDW methane emissions, the odds of case status also increased (OR=1.04, 95% CI: 1.00, 1.08). We found no association between PM2.5 or oil and gas wells and migraine case status. PM2.5 was linearly associated with neurology visits, migraine-specific urgent care visits, and MPA score>100, but not triptans or ED visits. NO2 was associated with migraine-specific urgent care and ED visits, but not other severity measures. We observed limited or null associations between continuous measures of methane emissions and proximity to oil and gas wells and migraine severity.CONCLUSIONS: Our findings illustrate the potential role of long-term exposure to multiple ambient air pollutants for prevalent migraine and migraine severity.

    View details for DOI 10.1186/s12940-021-00727-w

    View details for PubMedID 33865403

  • Mini-Review Free Sialic Acid Storage Disorder: Progress and Promise. Neuroscience letters Huizing, M., Hackbarth, M. E., Adams, D. R., Wasserstein, M., Patterson, M. C., Walkley, S. U., Gahl, W. A., FSASD Consortium, Adams, D. R., Dobrenis, K., Foglio, J., Gahl, W. A., Gasnier, B., Hackbarth, M., Huizing, M., Lek, M., Malicdan, M. C., Paavola, L. E., Patterson, M. C., Reimer, R., Walkley, S. U., Wasserstein, M., Wang, R. Y., Zoncu, R. 2021: 135896


    Lysosomal free sialic acid storage disorder (FSASD) is an extremely rare, autosomal recessive, neurodegenerative, multisystemic disorder caused by defects in the lysosomal sialic acid membrane exporter SLC17A5 (sialin). SLC17A5 defects cause free sialic acid and some other acidic hexoses to accumulate in lysosomes, resulting in enlarged lysosomes in some cell types and 10-100-fold increased urinary excretion of free sialic acid. Clinical features of FSASD include coarse facial features, organomegaly, and progressive neurodegenerative symptoms with cognitive impairment, cerebellar ataxia and muscular hypotonia. Central hypomyelination with cerebellar atrophy and thinning of the corpus callosum are also prominent disease features. Around 200 FSASD cases are reported worldwide, with the clinical spectrum ranging from a severe infantile onset form, often lethal in early childhood, to a mild, less severe form with subjects living into adulthood, also called Salla disease. The pathobiology of FSASD remains poorly understood and FSASD is likely underdiagnosed. Known patients have experienced a diagnostic delay due to the rarity of the disorder, absence of routine urine sialic acid testing, and non-specific clinical symptoms, including developmental delay, ataxia and infantile hypomyelination. There is no approved therapy for FSASD. We initiated a multidisciplinary collaborative effort involving worldwide academic clinical and scientific FSASD experts, the National Institutes of Health (USA), and the FSASD patient advocacy group (Salla Treatment and Research [S.T.A.R.] Foundation) to overcome the scientific, clinical and financial challenges facing the development of new treatments for FSASD. We aim to collect data that incentivize industry to further develop, obtain approval for, and commercialize FSASD treatments. This review summarizes current aspects of FSASD diagnosis, prevalence, etiology, and disease models, as well as challenges on the path to therapeutic approaches for FSASD.

    View details for DOI 10.1016/j.neulet.2021.135896

    View details for PubMedID 33862140

  • Air pollution, methane super-emitters, and oil and gas wells in Northern California: the relationship with migraine headache prevalence and exacerbation Elser, H., Morello-Frosch, R., Jacobson, A., Pressman, A., Kioumourtzoglou, M., Reimer, R., Casey, J. LIPPINCOTT WILLIAMS & WILKINS. 2021
  • Targeted genomic CRISPR-Cas9 screen identifies MAP4K4 as essential for glioblastoma invasion. Scientific reports Prolo, L. M., Li, A. n., Owen, S. F., Parker, J. J., Foshay, K. n., Nitta, R. T., Morgens, D. W., Bolin, S. n., Wilson, C. M., Vega L, J. C., Luo, E. J., Nwagbo, G. n., Waziri, A. n., Li, G. n., Reimer, R. J., Bassik, M. C., Grant, G. A. 2019; 9 (1): 14020


    Among high-grade brain tumors, glioblastoma is particularly difficult to treat, in part due to its highly infiltrative nature which contributes to the malignant phenotype and high mortality in patients. In order to better understand the signaling pathways underlying glioblastoma invasion, we performed the first large-scale CRISPR-Cas9 loss of function screen specifically designed to identify genes that facilitate cell invasion. We tested 4,574 genes predicted to be involved in trafficking and motility. Using a transwell invasion assay, we discovered 33 genes essential for invasion. Of the 11 genes we selected for secondary testing using a wound healing assay, 6 demonstrated a significant decrease in migration. The strongest regulator of invasion was mitogen-activated protein kinase 4 (MAP4K4). Targeting of MAP4K4 with single guide RNAs or a MAP4K4 inhibitor reduced migration and invasion in vitro. This effect was consistent across three additional patient derived glioblastoma cell lines. Analysis of epithelial-mesenchymal transition markers in U138 cells with lack or inhibition of MAP4K4 demonstrated protein expression consistent with a non-invasive state. Importantly, MAP4K4 inhibition limited migration in a subset of human glioma organotypic slice cultures. Our results identify MAP4K4 as a novel potential therapeutic target to limit glioblastoma invasion.

    View details for DOI 10.1038/s41598-019-50160-w

    View details for PubMedID 31570734

  • Bassoon Controls Presynaptic Autophagy through Atg5. Neuron Okerlund, N. D., Schneider, K., Leal-Ortiz, S., Montenegro-Venegas, C., Kim, S. A., Garner, L. C., Gundelfinger, E. D., Reimer, R. J., Garner, C. C. 2017; 93 (4): 897-913 e7


    Mechanisms regulating the surveillance and clearance of synaptic proteins are not well understood. Intriguingly, the loss of the presynaptic active zone proteins Piccolo and Bassoon triggers the loss of synaptic vesicles (SVs) and compromises synaptic integrity. Here we report that the destruction of SVs in boutons lacking Piccolo and Bassoon was associated with the induction of presynaptic autophagy, a process that depended on poly-ubiquitination, but not the E3 ubiquitin ligase Siah1. Surprisingly, gain or loss of function (LOF) of Bassoon alone suppressed or enhanced presynaptic autophagy, respectively, implying a fundamental role for Bassoon in the local regulation of presynaptic autophagy. Mechanistically, Bassoon was found to interact with Atg5, an E3-like ligase essential for autophagy, and to inhibit the induction of autophagy in heterologous cells. Importantly, Atg5 LOF as well as targeting an Atg5-binding peptide derived from Bassoon inhibited presynaptic autophagy in boutons lacking Piccolo and Bassoon, providing insights into the molecular mechanisms regulating presynaptic autophagy.

    View details for DOI 10.1016/j.neuron.2017.01.026

    View details for PubMedID 28231469

  • Human Astrocyte Maturation Captured in 3D Cerebral Cortical Spheroids Derived from Pluripotent Stem Cells. Neuron Sloan, S. A., Darmanis, S. n., Huber, N. n., Khan, T. A., Birey, F. n., Caneda, C. n., Reimer, R. n., Quake, S. R., Barres, B. A., Paşca, S. P. 2017; 95 (4): 779–90.e6


    There is significant need to develop physiologically relevant models for investigating human astrocytes in health and disease. Here, we present an approach for generating astrocyte lineage cells in a three-dimensional (3D) cytoarchitecture using human cerebral cortical spheroids (hCSs) derived from pluripotent stem cells. We acutely purified astrocyte-lineage cells from hCSs at varying stages up to 20 months in vitro using immunopanning and cell sorting and performed high-depth bulk and single-cell RNA sequencing to directly compare them to purified primary human brain cells. We found that hCS-derived glia closely resemble primary human fetal astrocytes and that, over time in vitro, they transition from a predominantly fetal to an increasingly mature astrocyte state. Transcriptional changes in astrocytes are accompanied by alterations in phagocytic capacity and effects on neuronal calcium signaling. These findings suggest that hCS-derived astrocytes closely resemble primary human astrocytes and can be used for studying development and modeling disease.

    View details for PubMedID 28817799

  • Trio, a Rho Family GEF, Interacts with the Presynaptic Active Zone Proteins Piccolo and Bassoon PLOS ONE Terry-Lorenzo, R. T., Torres, V. I., Wagh, D., Galaz, J., Swanson, S. K., Florens, L., Washburn, M. P., Waites, C. L., Gundelfinger, E. D., Reimer, R. J., Garner, C. C. 2016; 11 (12)


    Synaptic vesicles (SVs) fuse with the plasma membrane at a precise location called the presynaptic active zone (AZ). This fusion is coordinated by proteins embedded within a cytoskeletal matrix assembled at the AZ (CAZ). In the present study, we have identified a novel binding partner for the CAZ proteins Piccolo and Bassoon. This interacting protein, Trio, is a member of the Dbl family of guanine nucleotide exchange factors (GEFs) known to regulate the dynamic assembly of actin and growth factor dependent axon guidance and synaptic growth. Trio was found to interact with the C-terminal PBH 9/10 domains of Piccolo and Bassoon via its own N-terminal Spectrin repeats, a domain that is also critical for its localization to the CAZ. Moreover, our data suggest that regions within the C-terminus of Trio negatively regulate its interactions with Piccolo/Bassoon. These findings provide a mechanism for the presynaptic targeting of Trio and support a model in which Piccolo and Bassoon play a role in regulating neurotransmission through interactions with proteins, including Trio, that modulate the dynamic assembly of F-actin during cycles of synaptic vesicle exo- and endocytosis.

    View details for DOI 10.1371/journal.pone.0167535

    View details for Web of Science ID 000389482700187

    View details for PubMedID 27907191

    View details for PubMedCentralID PMC5132261

  • Plasma taurine levels are not affected by vigabatrin in pediatric patients. Epilepsia Spelbrink, E. M., Mabud, T. S., Reimer, R., Porter, B. E. 2016; 57 (8): e168-72


    Vigabatrin is a highly effective antiseizure medication, but its use is limited due to concerns about retinal toxicity. One proposed mechanism for this toxicity is vigabatrin-mediated reduction of taurine. Herein we assess plasma taurine levels in a retrospective cohort of children with epilepsy, including a subset receiving vigabatrin. All children who underwent a plasma amino acid analysis as part of their clinical evaluation between 2006 and 2015 at Stanford Children's Health were included in the analysis. There were no significant differences in plasma taurine levels between children taking vigabatrin (n = 16), children taking other anti-seizure medications, and children not taking any anti-seizure medication (n = 556) (analysis of variance [ANOVA] p = 0.841). There were, however, age-dependent decreases in plasma taurine levels. Multiple linear regression revealed no significant association between vigabatrin use and plasma taurine level (p = 0.87) when controlling for age. These results suggest that children taking vigabatrin maintain normal plasma taurine levels, although they leave unanswered whether taurine supplementation is necessary or sufficient to prevent vigabatrin-associated visual field loss. They also indicate that age should be taken into consideration when evaluating taurine levels in young children.

    View details for DOI 10.1111/epi.13447

    View details for PubMedID 27344989

  • Piccolo Directs Activity Dependent F-Actin Assembly from Presynaptic Active Zones via Daam1 PLOS ONE Wagh, D., Terry-Lorenzo, R., Waites, C. L., Leal-Ortiz, S. A., Maas, C., Reimer, R. J., Garner, C. C. 2015; 10 (4)


    The dynamic assembly of filamentous (F) actin plays essential roles in the assembly of presynaptic boutons, the fusion, mobilization and recycling of synaptic vesicles (SVs), and presynaptic forms of plasticity. However, the molecular mechanisms that regulate the temporal and spatial assembly of presynaptic F-actin remain largely unknown. Similar to other F-actin rich membrane specializations, presynaptic boutons contain a set of molecules that respond to cellular cues and trans-synaptic signals to facilitate activity-dependent assembly of F-actin. The presynaptic active zone (AZ) protein Piccolo has recently been identified as a key regulator of neurotransmitter release during SV cycling. It does so by coordinating the activity-dependent assembly of F-Actin and the dynamics of key plasticity molecules including Synapsin1, Profilin and CaMKII. The multidomain structure of Piccolo, its exquisite association with the AZ, and its ability to interact with a number of actin-associated proteins suggest that Piccolo may function as a platform to coordinate the spatial assembly of F-actin. Here we have identified Daam1, a Formin that functions with Profilin to drive F-actin assembly, as a novel Piccolo binding partner. We also found that within cells Daam1 activation promotes Piccolo binding, an interaction that can spatially direct the polymerization of F-Actin. Moreover, similar to Piccolo and Profilin, Daam1 loss of function impairs presynaptic-F-actin assembly in neurons. These data suggest a model in which Piccolo directs the assembly of presynaptic F-Actin from the AZ by scaffolding key actin regulatory proteins including Daam1.

    View details for DOI 10.1371/journal.pone.0120093

    View details for Web of Science ID 000353212600003

    View details for PubMedID 25897839

    View details for PubMedCentralID PMC4405365

  • Endozepines. Advances in pharmacology (San Diego, Calif.) Farzampour, Z., Reimer, R. J., Huguenard, J. 2015; 72: 147-164


    Since their introduction in the 1960s, benzodiazepines (BZs) remain one of the most commonly prescribed medications, acting as potent sedatives, hypnotics, anxiolytics, anticonvulsants, and muscle relaxants. The primary neural action of BZs and related compounds is augmentation of inhibitory transmission, which occurs through allosteric modulation of the gamma-aminobutyric acid (GABA)-induced current at the gamma-aminobutyric acid receptor (GABAAR). The discovery of the BZ-binding site on GABAARs encouraged many to speculate that the brain produces its own endogenous ligands to this site (Costa & Guidotti, 1985). The romanticized quest for endozepines, endogenous ligands to the BZ-binding site, has uncovered a variety of ligands that might fulfill this role, including oleamides (Cravatt et al., 1995), nonpeptidic endozepines (Rothstein et al., 1992), and the protein diazepam-binding inhibitor (DBI) (Costa & Guidotti, 1985). Of these ligands, DBI, and affiliated peptide fragments, is the most extensively studied endozepine. The quest for the "brain's Valium" over the decades has been elusive as mainly negative allosteric modulatory effects have been observed (Alfonso, Le Magueresse, Zuccotti, Khodosevich, & Monyer, 2012; Costa & Guidotti, 1985), but recent evidence is accumulating that DBI displays regionally discrete endogenous positive modulation of GABA transmission through activation of the BZ receptor (Christian et al., 2013). Herein, we review the literature on this topic, focusing on identification of the endogenous molecule and its region-specific expression and function.

    View details for DOI 10.1016/bs.apha.2014.10.005

    View details for PubMedID 25600369

  • alpha 5-GABAA receptors negatively regulate MYC-amplified medulloblastoma growth ACTA NEUROPATHOLOGICA Sengupta, S., Weeraratne, S. D., Sun, H., Phallen, J., Rallapalli, S. K., Teider, N., Kosaras, B., Amani, V., Pierre-Francois, J., Tang, Y., Brian Nguyen, B., Yu, F., Schubert, S., Balansay, B., Mathios, D., Lechpammer, M., Archer, T. C., Phuoc Tran, P., Reimer, R. J., Cook, J. M., Lim, M., Jensen, F. E., Pomeroy, S. L., Cho, Y. 2014; 127 (4): 593-603


    Neural tumors often express neurotransmitter receptors as markers of their developmental lineage. Although these receptors have been well characterized in electrophysiological, developmental and pharmacological settings, their importance in the maintenance and progression of brain tumors and, importantly, the effect of their targeting in brain cancers remains obscure. Here, we demonstrate high levels of GABRA5, which encodes the α5-subunit of the GABAA receptor complex, in aggressive MYC-driven, "Group 3" medulloblastomas. We hypothesized that modulation of α5-GABAA receptors alters medulloblastoma cell survival and monitored biological and electrophysiological responses of GABRA5-expressing medulloblastoma cells upon pharmacological targeting of the GABAA receptor. While antagonists, inverse agonists and non-specific positive allosteric modulators had limited effects on medulloblastoma cells, a highly specific and potent α5-GABAA receptor agonist, QHii066, resulted in marked membrane depolarization and a significant decrease in cell survival. This effect was GABRA5 dependent and mediated through the induction of apoptosis as well as accumulation of cells in S and G2 phases of the cell cycle. Chemical genomic profiling of QHii066-treated medulloblastoma cells confirmed inhibition of MYC-related transcriptional activity and revealed an enrichment of HOXA5 target gene expression. siRNA-mediated knockdown of HOXA5 markedly blunted the response of medulloblastoma cells to QHii066. Furthermore, QHii066 sensitized GABRA5 positive medulloblastoma cells to radiation and chemotherapy consistent with the role of HOXA5 in directly regulating p53 expression and inducing apoptosis. Thus, our results provide novel insights into the synthetic lethal nature of α5-GABAA receptor activation in MYC-driven/Group 3 medulloblastomas and propose its targeting as a novel strategy for the management of this highly aggressive tumor.

    View details for DOI 10.1007/s00401-013-1205-7

    View details for Web of Science ID 000332957400010

  • A local glutamate-glutamine cycle sustains synaptic excitatory transmitter release. Neuron Tani, H., Dulla, C. G., Farzampour, Z., Taylor-Weiner, A., Huguenard, J. R., Reimer, R. J. 2014; 81 (4): 888-900


    Biochemical studies suggest that excitatory neurons are metabolically coupled with astrocytes to generate glutamate for release. However, the extent to which glutamatergic neurotransmission depends on this process remains controversial because direct electrophysiological evidence is lacking. The distance between cell bodies and axon terminals predicts that glutamine-glutamate cycle is synaptically localized. Hence, we investigated isolated nerve terminals in brain slices by transecting hippocampal Schaffer collaterals and cortical layer I axons. Stimulating with alternating periods of high frequency (20 Hz) and rest (0.2 Hz), we identified an activity-dependent reduction in synaptic efficacy that correlated with reduced glutamate release. This was enhanced by inhibition of astrocytic glutamine synthetase and reversed or prevented by exogenous glutamine. Importantly, this activity dependence was also revealed with an in-vivo-derived natural stimulus both at network and cellular levels. These data provide direct electrophysiological evidence that an astrocyte-dependent glutamate-glutamine cycle is required to maintain active neurotransmission at excitatory terminals.

    View details for DOI 10.1016/j.neuron.2013.12.026

    View details for PubMedID 24559677

  • SLC17: a functionally diverse family of organic anion transporters. Molecular aspects of medicine Reimer, R. J. 2013; 34 (2-3): 350-359


    Molecular studies have determined that the SLC17 transporters, a family of nine proteins initially implicated in phosphate transport, 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 neurotransmitters, to urate metabolism, to the degradation and metabolism of glycoproteins.

    View details for DOI 10.1016/j.mam.2012.05.004

    View details for PubMedID 23506876

  • Vesicular uptake and exocytosis of L-aspartate is independent of sialin FASEB JOURNAL Morland, C., Nordengen, K., Larsson, M., Prolo, L. M., Farzampour, Z., Reimer, R. J., Gundersen, V. 2013; 27 (3): 1264-1274


    The mechanism of release and the role of l-aspartate as a central neurotransmitter are controversial. A vesicular release mechanism for l-aspartate has been difficult to prove, as no vesicular l-aspartate transporter was identified until it was found that sialin could transport l-aspartate and l-glutamate when reconstituted into liposomes. We sought to clarify the release mechanism of l-aspartate and the role of sialin in this process by combining l-aspartate uptake studies in isolated synaptic vesicles with immunocyotchemical investigations of hippocampal slices. We found that radiolabeled l-aspartate was taken up into synaptic vesicles. The vesicular l-aspartate uptake, relative to the l-glutamate uptake, was twice as high in the hippocampus as in the whole brain, the striatum, and the entorhinal and frontal cortices and was not inhibited by l-glutamate. We further show that sialin is not essential for exocytosis of l-aspartate, as there was no difference in ATP-dependent l-aspartate uptake in synaptic vesicles from sialin-knockout and wild-type mice. In addition, expression of sialin in PC12 cells did not result in significant vesicle uptake of l-aspartate, and depolarization-induced depletion of l-aspartate from hippocampal nerve terminals was similar in hippocampal slices from sialin-knockout and wild-type mice. Further, there was no evidence for nonvesicular release of l-aspartate via volume-regulated anion channels or plasma membrane excitatory amino acid transporters. This suggests that l-aspartate is exocytotically released from nerve terminals after vesicular accumulation by a transporter other than sialin.

    View details for DOI 10.1096/fj.12-206300

    View details for Web of Science ID 000315585200038

    View details for PubMedID 23221336

    View details for PubMedCentralID PMC3574276

  • Glutamate biosensor imaging reveals dysregulation of glutamatergic pathways in a model of developmental cortical malformation NEUROBIOLOGY OF DISEASE Dulla, C. G., Tani, H., Brill, J., Reimer, R. J., Huguenard, J. R. 2013; 49: 232-246
  • Glutamate biosensor imaging reveals dysregulation of glutamatergic pathways in a model of developmental cortical malformation. Neurobiology of disease Dulla, C. G., Tani, H., Brill, J., Reimer, R. J., Huguenard, J. R. 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 DOI 10.1016/j.nbd.2012.09.001

    View details for PubMedID 22982711

    View details for PubMedCentralID PMC3574209

  • Biochemistry to the Rescue: A CIC-2 Auxiliary Subunit Provides a Tangible Link to Leukodystrophy NEURON Maduke, M. C., Reimer, R. J. 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

  • Structure-Function Studies of the SLC17 Transporter Sialin Identify Crucial Residues and Substrate-induced Conformational Changes JOURNAL OF BIOLOGICAL CHEMISTRY Courville, P., Quick, M., Reimer, R. J. 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

    View details for PubMedCentralID PMC2885210

  • Glutamine Is Required for Persistent Epileptiform Activity in the Disinhibited Neocortical Brain Slice JOURNAL OF NEUROSCIENCE Tani, H., Dulla, C. G., Huguenard, J. R., Reimer, R. J. 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

    View details for PubMedCentralID PMC2821093

  • The Lysosomal Sialic Acid Transporter Sialin Is Required for Normal CNS Myelination JOURNAL OF NEUROSCIENCE Prolo, L. M., Vogel, H., Reimer, R. J. 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 PubMedID 20007460

  • Synaptic Vesicle Protein NTT4/XT1 (SLC6A17) Catalyzes Na+-coupled Neutral Amino Acid Transport JOURNAL OF BIOLOGICAL CHEMISTRY Zaia, K. A., Reimer, R. J. 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

    View details for PubMedCentralID PMC2659202

  • Imaging of glutamate in brain slices using FRET sensors JOURNAL OF NEUROSCIENCE METHODS Dulla, C., Tani, H., Okumoto, S., Frommer, W. B., Reimer, R. J., Fluguenard, J. R. 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

    View details for PubMedCentralID PMC2267481

  • G328E and G409E sialin missense mutations similarly impair transport activity, but differentially affect trafficking MOLECULAR GENETICS AND METABOLISM Myall, N. J., Wreden, C. C., Wlizla, M., Reimer, R. J. 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

    View details for PubMedCentralID PMC2171360

  • Analysis of glutamate recycling in maintenance of epileptiform activity the acute slice Reimer, R. J., Tani, H., Dulla, C. G., Huguenard, J. R. BLACKWELL PUBLISHING. 2007: 240
  • Membrane topology of the Drosophila vesicular glutamate transporter JOURNAL OF NEUROCHEMISTRY Fei, H., Karnezis, T., Reimer, R. J., Krantz, D. E. 2007; 101 (6): 1662-1671


    The vesicular glutamate transporters (VGLUTs) are responsible for packaging glutamate into synaptic vesicles, and are part of a family of structurally related proteins that mediate organic anion transport. Standard computer-based predictions of transmembrane domains have led to divergent topological models, indicating the need for experimentally derived predictions. Here we present data on the topology of the VGLUT ortholog from Drosophila melanogaster (DVGLUT). Using immunofluorescence assays of DVGLUT transiently localized to the plasma membrane of heterologously transfected cells, we have determined the accessibility of epitope tags inserted into the lumenal/extracellular face of the protein. Using immunoisolation, we have identified complementary tagged sites that face the cytoplasm. Our data show that DVGLUT contains 10 hydrophobic regions that completely span the membrane (TMs 1-10) and that the amino and carboxyl termini are cytosolic. Importantly, between TMs 4 and 5 is an unforeseen cytosolic loop of some 50 residues. Other domains exposed to the cytosol include loops between TMs 6-7 and 8-9, and regions C-terminal to TM2 and N-terminal to TM3. Between TM2 and 3 is a potentially hydrophobic, but topologically ambiguous region. Lumenal domains include sequences between TMs 1-2, 3-4, 5-6, 7-8 and 9-10. These data provide a basis for determining structure-function relationships for DVGLUT and other related proteins.

    View details for DOI 10.1111/j.1471-4159.2007.04518.x

    View details for Web of Science ID 000247135300020

    View details for PubMedID 17394549

  • Modulation of epileptiform activity by glutamine and system A transport in a model of post-traumatic epilepsy NEUROBIOLOGY OF DISEASE Tani, H., Bandrowski, A. E., Parada, I., Wynn, M., Huguenard, J. R., Prince, D. A., Reimer, R. J. 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

    View details for PubMedCentralID PMC1952182

  • Biochemical and genetic analysis of ANK in arthritis and bone disease AMERICAN JOURNAL OF HUMAN GENETICS Gurley, K. A., Reimer, R. J., Kingsley, D. M. 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 Okumoto, S., Looger, L. L., Micheva, K. D., Reimer, R. J., Smith, S. J., Frommer, W. B. 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

    View details for PubMedCentralID PMC1143584

  • Varied mechanisms underlie the free sialic acid storage disorders JOURNAL OF BIOLOGICAL CHEMISTRY Wreden, C. C., Wlizla, M., Reimer, R. J. 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 Reimer, R. J., Edwards, R. H. 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

  • Vesicular neurotransmitter transporter expression in developing postnatal rodent retina: GABA and glycine precede glutamate JOURNAL OF NEUROSCIENCE Johnson, J., Tian, N., Caywood, M. S., Reimer, R. J., Edwards, R. H., Copenhagen, D. R. 2003; 23 (2): 518–29


    Vesicular transporters regulate the amount and type of neurotransmitter sequestered into synaptic vesicles and, hence, the kind of signal transmitted to postsynaptic neurons. Glutamate is the prominent excitatory neurotransmitter in retina; GABA and glycine are the main inhibitory neurotransmitters. Little is known about the ontogeny of vesicular neurotransmission in retina. We investigated expression of glutamatergic [vesicular glutamate transporter 1 (VGLUT1)] and GABA/glycinergic [vesicular GABA/glycine transporter (VGAT)] vesicular transporters in postnatal retina. VGLUT1 labels glutamatergic synapses. VGLUT1 and synaptic vesicle 2 colocalized to photoreceptor terminals. VGLUT1 colocalized with PKC to rod bipolar terminals and to ON bipolar terminals in metabotropic glutamate receptor 6+/- mice. Developmentally, VGAT expression precedes VGLUT1. In rat and mouse retina, VGAT occurred in the inner retina by postnatal day 1 (P1). In rat retina, VGLUT1 was in the outer retina by P5-P7 and the inner retina by P7. In the mouse retina, VGLUT1 expression was in the outer retina by P3 and the inner retina by P5. Both rat and mouse retina had an adult pattern of VGLUT1 expression by P14. VGLUT1 expression precedes ribbon synapses, which are first observed in the inner retina at P11 (Fisher, 1979) in mouse and P13 (Horsburgh and Sefton, 1987) in rat. The ribbon synapse marker RIBEYE was not detected in inner retina of P5 or P7 rat. Spontaneous EPSCs in mouse ganglion cells were recorded as early as P7. Together, these findings indicate that vesicular GABA and glycine transmission precedes vesicular glutamate transmission in developing rodent retina. Furthermore, vesicular glutamate transmission likely occurs before ribbon synapse formation in the inner retina.

    View details for Web of Science ID 000180523000018

    View details for PubMedID 12533612