Tara M. DeSilva

LRRK2 Inhibition Attenuates Microglial Inflammatory Responses

Missense mutations in leucine-rich repeat kinase 2 (LRRK2) cause late-onset Parkinsons disease (PD), and common genetic variation in LRRK2 modifies susceptibility to Crohns disease and leprosy. High levels of LRRK2 expression in peripheral monocytes and macrophages suggest a role for LRRK2 in these cells, yet little is known about LRRK2 expression and function in immune cells of the brain. Here, we demonstrate a role for LRRK2 in mediating microglial proinflammatory responses and morphology. In a murine model of neuroinflammation, we observe robust induction of LRRK2 in microglia. Experiments with toll-like receptor 4 (TLR4)-stimulated rat primary microglia show that inflammation increases LRRK2 activity and expression, while inhibition of LRRK2 kinase activity or knockdown of protein attenuates TNFα secretion and nitric oxide synthase (iNOS) induction. LRRK2 inhibition blocks TLR4 stimulated microglial process outgrowth and impairs ADP stimulated microglial chemotaxis. However, actin inhibitors that phenocopy inhibition of process outgrowth and chemotaxis fail to modify TLR4 stimulation of TNFα secretion and inducible iNOS induction, suggesting that LRRK2 acts upstream of cytoskeleton control as a stress-responsive kinase. These data demonstrate LRRK2 in regulating responses in immune cells of the brain and further implicate microglial involvement in late-onset PD.

The Glutamate Transporter GLT1a Is Expressed in Excitatory Axon Terminals of Mature Hippocampal Neurons

GLT1 is the major glutamate transporter of the brain and has been thought to be expressed exclusively in astrocytes. Although excitatory axon terminals take up glutamate, the transporter responsible has not been identified. GLT1 is expressed in at least two forms varying in the C termini, GLT1a and GLT1b. GLT1 mRNA has been demonstrated in neurons, without associated protein. Recently, evidence has been presented, using specific C terminus-directed antibodies, that GLT1b protein is expressed in neurons in vivo. These data suggested that the GLT1 mRNA detected in neurons encodes GLT1b and also that GLT1b might be the elusive presynaptic transporter. To test these hypotheses, we used variant-specific probes directed to the 3′-untranslated regions for GLT1a and GLT1b to perform in situ hybridization in the hippocampus. Contrary to expectation, GLT1a mRNA was the more abundant form. To investigate further the expression of GLT1 in neurons in the hippocampus, antibodies raised against the C terminus of GLT1a and against the N terminus of GLT1, found to be specific by testing in GLT1 knock-out mice, were used for light microscopic and EM-ICC. GLT1a protein was detected in neurons, in 14–29% of axons in the hippocampus, depending on the region. Many of the labeled axons formed axo-spinous, asymmetric, and, thus, excitatory synapses. Labeling also occurred in some spines and dendrites. The antibody against the N terminus of GLT1 also produced labeling of neuronal processes. Thus, the originally cloned form of GLT1, GLT1a, is expressed as protein in neurons in the mature hippocampus and may contribute significantly to glutamate uptake into excitatory terminals.

Axonal development in the cerebral white matter of the human fetus and infant

After completion of neuronal migration to form the cerebral cortex, axons undergo rapid elongation to their intra‐ and subcortical targets, from midgestation through infancy. We define axonal development in the human parietal white matter in this critical period. Immunocytochemistry and Western blot analysis were performed on 46 normative cases from 20–183 postconceptional (PC) weeks. Anti‐SMI 312, a pan‐marker of neurofilaments, stained axons as early as 23 weeks. Anti‐SMI 32, a marker for nonphosphorylated neurofilament high molecular weight (NFH), primarily stained neuronal cell bodies (cortical, subcortical, and Cajal‐Retzius). Anti‐SMI 31, which stains phosphorylated NFH, was used as a marker of axonal maturity, and showed relatively low levels of staining (approximately one‐fourth of adult levels) from 24–34 PC weeks. GAP‐43, a marker of axonal growth and elongation, showed high levels of expression in the white matter from 21–64 PC weeks and lower, adult‐like levels beyond 17 postnatal months. The onset of myelination, as seen by myelin basic protein expression, was ∼54 weeks, with progression to “adult‐like” staining by 72–92 PC weeks. This study provides major insight into axonal maturation during a critical period of growth, over an age range not previously examined and one coinciding with the peak period of periventricular leukomalacia (PVL), the major disorder underlying cerebral palsy in premature infants. These data suggest that immature axons are susceptible to damage in PVL and that the timing of axonal maturation must be considered toward establishing its pathology relative to the oligodendrocyte/myelin/axonal unit. J. Comp. Neurol. 484:156–167, 2005.

Hyperoxia Causes Maturation-Dependent Cell Death in the Developing White Matter

Periventricular leukomalacia is the predominant injury in the preterm infant leading to cerebral palsy. Oxygen exposure may be an additional cause of brain injury in these infants. In this study, we investigated pathways of maturation-dependent oligodendrocyte (OL) death induced by hyperoxia in vitro and in vivo. Developing and mature OLs were subjected to 80% oxygen (0–24 h). Lactate dehydrogenase (LDH) assay was used to assess cell viability. Furthermore, 3-, 6-, and 10-d-old rat pups were subjected to 80% oxygen (24 h), and their brains were processed for myelin basic protein staining. Significant cell death was detected after 6–24 h incubation in 80% oxygen in pre-OLs (O4+,O1−), but not in mature OLs (MBP+). Cell death was executed by a caspase-dependent apoptotic pathway and could be blocked by the pan-caspase inhibitor zVAD-fmk. Overexpression of BCL2 (Homo sapiens B-cell chronic lymphocytic leukemia/lymphoma 2) significantly reduced apoptosis. Accumulation of superoxide and generation of reactive oxygen species (ROS) were detected after 2 h of oxygen exposure. Lipoxygenase inhibitors 2,3,5-trimethyl-6-(12-hydroxy-5-10-dodecadiynyl-1,4-benzoquinone and N-benzyl-N-hydroxy-5-phenylpentamide fully protected the cells from oxidative injury. Overexpression of superoxide dismutase (SOD1) dramatically increased injury to pre-OLs but not to mature OLs. We extended these studies by testing the effects of hyperoxia on neonatal white matter. Postnatal day 3 (P3) and P6 rats, but not P10 pups, showed bilateral reduction in MBP (myelin basic protein) expression with 24 h exposure to 80% oxygen. Hyperoxia causes oxidative stress and triggers maturation-dependent apoptosis in pre-OLs, which involves the generation of ROS and caspase activation, and leads to white matter injury in the neonatal rat brain. These observations may be relevant to white matter injury observed in premature infants.

Cellular and Subcellular mRNA Localization of Glutamate Transporter Isoforms GLT1a and GLT1b in Rat Brain by In Situ Hybridization

GLT1, the predominant glutamate transporter of the forebrain, exists in two splice variant isoforms, i.e., GLT1a and GLT1b. Although GLT1 was originally detected only in astrocytes, we have recently demonstrated that GLT1a protein is expressed by neurons in the hippocampus as well. In the present study, the mRNA distribution patterns for the two isoforms were examined throughout the rat brain by using nonisotopic in situ hybridization and variant‐specific RNA probes. Both isoforms were expressed in neuronal subgroups outside the hippocampus, such as in the cerebral cortex layer VI, or the neurons in the olfactory tubercle. As was the case in the hippocampus, GLT1a was the predominant transcript in neurons in these regions as well. Both GLT1 isoforms were widely expressed in astrocytes throughout the brain. GLT1a mRNA expression in astrocytes showed noticeable variation in labeling intensity in subregions of the hippocampus and other areas, whereas GLT1b expression in astrocytes was relatively homogeneous. On the subcellular level, GLT1a mRNA was expressed primarily in astrocyte processes, whereas GLT1b mRNA was more restricted to the astrocyte cell body. The two isoforms showed similar distributions in the subfornical organ and in tanycytes of the third ventricle. However, GLT1 expression in the pineal gland and the retina was due primarily to GLT1b, whereas GLT1a was more strongly expressed in Bergman glia in the cerebellum. These findings suggest that the expression of the two GLT1 isoforms is regulated by different mechanisms. Moreover, the function of the two isoforms may be subject to different regulatory processes. J. Comp. Neurol. 492:78–89, 2005.

Overexpression, purification, and characterization of recombinant Ca-ATPase regulators for high-resolution solution and solid-state NMR studies.

Phospholamban (PLB) and Sarcolipin (SLN) are integral membrane proteins that regulate muscle contractility via direct interaction with the Ca-ATPase in cardiac and skeletal muscle, respectively. The molecular details of these protein-protein interactions are as yet undetermined. Solution and solid-state NMR spectroscopies have proven to be effective tools for deciphering such regulatory mechanisms to a high degree of resolution; however, large quantities of pure recombinant protein are required for these studies. Thus, recombinant PLB and SLN production in Escherichia coli was optimized for use in NMR experiments. Fusions of PLB and SLN to maltose binding protein (MBP) were constructed and optimal conditions for protein expression and purification were screened. This facilitated the large-scale production of highly pure protein. To confirm their functionality, the biological activities of recombinant PLB and SLN were compared to those of their synthetic counterparts. The regulation of Ca-ATPase activity by recombinant PLB and SLN was indistinguishable from the regulation by synthetic proteins, demonstrating the functional integrity of the recombinant constructs and ensuring the biological relevance of our future structural studies. Finally, NMR spectroscopic conditions were established and optimized for use in investigations of the mechanism of Ca-ATPase regulation by PLB and SLN.

17β-Estradiol Protects Against Hypoxic/Ischemic White Matter Damage in the Neonatal Rat Brain

Developing oligodendrocytes (pre‐OLs) are highly vulnerable to hypoxic‐ischemic injury and associated excitotoxicity and oxidative stress. 17β‐Estradiol plays an important role in the development and function of the CNS and is neuroprotective. The sudden drop in circulating estrogen after birth may enhance the susceptibility of developing OLs to injury. Estrogen receptor (ER)–α and ER‐β are both expressed in OLs. We examined the effect of 17β‐estradiol on oxygen‐glucose deprivation and oxidative stress–induced cell death in rat pre‐OLs in vitro and on hypoxic‐ischemic brain injury in vivo. Pre‐OLs in culture were subjected to oxygen‐glucose deprivation (OGD) or glutathione depletion in the presence or absence of 17β‐estradiol. LDH release, the Alamar blue assay, and phase‐contrast microscopy were used to assess cell viability. Hypoxic‐ischemic injury was generated in 6‐day‐old rats (P6) by unilateral carotid ligation and hypoxia (6% O2 for 1 hr). Rat pups received one intraperitoneal injection of 300 or 600 μg/kg 17β‐estradiol or vehicle 12 hr prior to the surgical procedure. Injury was assessed by myelin basic protein (MBP) immunocytochemistry at P10. 17β‐Estradiol produced significant protection against OGD‐induced cell death in primary OLs (EC50 = 1.3 ± 0.46 × 10−9 M) and against oxidative stress. Moreover, 17β‐estradiol attenuated the loss of MBP labeling in P10 pups ipsilateral to the carotid ligation. These results suggest a potential role for estrogens in attenuation of hypoxic‐ischemic and oxidative injury to developing OLs and in the prevention of periventricular leukomalacia.

The glutamate transporter EAAT2 is transiently expressed in developing human cerebral white matter

The major brain abnormality underlying cerebral palsy in premature infants is periventricular leukomalacia (PVL), a lesion of the immature cerebral white matter. Oligodendrocyte precursors (pre‐OLs; O4+O1−) predominate in human cerebral white matter during the peak time frame for PVL (24–32 gestational weeks) and are vulnerable to excitotoxicity. We hypothesize that PVL reflects, in part, excitotoxicity to pre‐OLs resulting from cerebral ischemia/reperfusion. Reversal of glutamate transport in the setting of energy failure is a major source of pathologic accumulation of extracellular glutamate. Here, we identify and localize the glutamate transporters in human cerebral white matter during the age range of PVL. In situ hybridization was performed with digoxigenin‐labeled probes directed against the full‐length coding regions of EAAT1, EAAT2, and EAAT3. EAAT2 mRNA was abundant in human fetal white matter during the period of peak incidence of PVL and virtually disappeared by 2 postnatal months. Its developmental profile differed significantly from that of both EAAT1 and EAAT3 mRNA. Immunoblotting demonstrated that EAAT2 protein was highly expressed in early development relative to adult values. Double‐label immunocytochemistry detected EAAT2 in OLs but not astrocytes or axons in the human fetal white matter. We conclude that transient expression of EAAT2 occurs during the window of peak vulnerability for PVL, suggesting that this developmentally up‐regulated transporter may be a major source of extracellular glutamate in ischemic injury to the cerebral white matter of the preterm infant. J. Comp. Neurol. 501:879–890, 2007.

Structural biology of metal-binding sequences

NMR spectroscopy and X-ray crystallography in conjunction with extended X-ray absorption fine structure spectroscopy, have contributed to the elucidation of the structural biology of protein-mediated mechanisms of heavy metal homeostasis. Among the most striking aspects of these investigations are the remarkable similarity of metal-ion-transport and sequestering systems across different species, and the need to continue the research to confirm hypotheses for the molecular mechanisms of transfers of metal ions between proteins.

Interaction between the glutamate transporter GLT1b and the synaptic PDZ domain protein PICK1

Synaptic plasticity is implemented by the interaction of glutamate receptors with PDZ domain proteins. Glutamate transporters provide the only known mechanism of clearance of glutamate from excitatory synapses, and GLT1 is the major glutamate transporter. We show here that GLT1 interacts with the PDZ domain protein PICK1, which plays a critical role in regulating the expression of glutamate receptors at excitatory synapses. A yeast two‐hybrid screen of a neuronal library using the carboxyl tail of GLT1b yielded clones expressing PICK1. The GLT1b C‐terminal peptide bound to PICK1 with high affinity (Ki = 6.5 ± 0.4 µm) in an in vitro fluorescence polarization assay. We also tested peptides based on other variants of GLT1 and other glutamate transporters. GLT1b co‐immunoprecipitated with PICK1 from rat brain lysates and COS7 cell lysates derived from cells transfected with plasmids expressing PICK1 and GLT1b. In addition, expression of GLT1b in COS7 cells changed the distribution of PICK1, bringing it to the surface. GLT1b and PICK1 co‐localized with each other and with synaptic markers in hippocampal neurons in culture. Phorbol ester, an activator of protein kinase C (PKC), a known PICK1 interactor, had no effect on glutamate transport in rat forebrain neurons in culture. However, we found that exposure of neurons to a myristolated decoy peptide with sequence identical to the C‐terminal sequence of GLT1b designed to block the PICK1–GLT1b interaction rendered glutamate transport into neurons responsive to phorbol ester. These results suggest that the PICK1–GLT1b interaction regulates the modulation of GLT1 function by PKC.