Dl Choi-Lundberg

Mice lacking α-synuclein display functional deficits in the nigrostriatal dopamine system

alpha-Synuclein (alpha-Syn) is a 14 kDa protein of unknown function that has been implicated in the pathophysiology of Parkinsons disease (PD). Here, we show that alpha-Syn-/- mice are viable and fertile, exhibit intact brain architecture, and possess a normal complement of dopaminergic cell bodies, fibers, and synapses. Nigrostriatal terminals of alpha-Syn-/- mice display a standard pattern of dopamine (DA) discharge and reuptake in response to simple electrical stimulation. However, they exhibit an increased release with paired stimuli that can be mimicked by elevated Ca2+. Concurrent with the altered DA release, alpha-Syn-/- mice display a reduction in striatal DA and an attenuation of DA-dependent locomotor response to amphetamine. These findings support the hypothesis that alpha-Syn is an essential presynaptic, activity-dependent negative regulator of DA neurotransmission.

Ontogeny and distribution of glial cell line-derived neurotrophic factor (GDNF) mRNA in rat

Glial cell line-derived neurotrophic factor (GDNF) is a member of the transforming growth factor-beta family isolated from the rat glial tumor cell line, B49. In embryonic dopaminergic (DA) neurons in vitro, GDNF promotes survival, high-affinity dopamine uptake, and neurite outgrowth. We have used a semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) with primers specific to GDNF to study the developmental expression of GDNF mRNA in central nervous system (CNS) and peripheral organs of embryonic rat on gestational days E11.5, E13.5 and E18, neonatal rat on postnatal days P0 and P10, and adult rat. GDNF mRNA is expressed throughout the CNS, with highest levels in P0 spinal cord and in P0 and P10 striatum. Lower levels are present in the brainstem (including the ventral mesencephalon, which contains the DA neurons of the substantia nigra), cerebellum, diencephalon, and telencephalon, as well as in primary cultures of cerebellar granule cells prepared from P7 cerebellum and astrocytes prepared from P1 cortex. The cerebellum has an unusual temporal pattern of expression, high at birth and in the adult, but undetectable at P10. GDNF mRNA is also expressed in many peripheral tissues at higher levels than in brain. These include embryonic limb bud, kidney and gut; neonatal kidney, gut, lung and testis; and adult lung, liver and ovary. In addition to the predicted RT-PCR product, we also observed a minor band which was shown to be identical to GDNF in the mature peptide sequence, but which has a 78 base pair deletion in the preproprotein sequence.(ABSTRACT TRUNCATED AT 250 WORDS)

Behavioral and cellular protection of rat dopaminergic neurons by an adenoviral vector encoding glial cell line-derived neurotrophic factor.

Previously, we observed that an adenoviral (Ad) vector encoding human glial cell line-derived neurotrophic factor (GDNF), injected near the rat substantia nigra (SN), protects SN dopaminergic (DA) neuronal soma from 6-hydroxydopamine (6-OHDA)-induced degeneration. In the present study, the effects of Ad GDNF injected into the striatum, the site of DA nerve terminals, were assessed in the same lesion model. So that effects on cell survival could be assessed without relying on DA phenotypic markers, fluorogold (FG) was infused bilaterally into striatae to retrogradely label DA neurons. Ad GDNF or control treatment (Ad mGDNF, encoding a deletion mutant GDNF, Ad lacZ, vehicle, or no injection) was injected unilaterally into the striatum near one FG site. Progressive degeneration of DA neurons was initiated 7 days later by unilateral injection of 6-OHDA at this FG site. At 42 days after 6-OHDA, Ad GDNF prevented the death of 40% of susceptible DA neurons that projected to the lesion site. Ad GDNF prevented the development of behavioral asymmetries which depend on striatal dopamine, including limb use asymmetries during spontaneous movements along vertical surfaces and amphetamine-induced rotation. Both behavioral asymmetries were exhibited by control-treated, lesioned rats. Interestingly, these behavioral protections occurred in the absence of an increase in the density of DA nerve fibers in the striatum of Ad GDNF-treated rats. ELISA measurements of transgene proteins showed that nanogram quantities of GDNF and lacZ transgene were present in the striatum for 7 weeks, and picogram quantities of GDNF in the SN due to retrograde transport of vector and/or transgene protein. These studies demonstrate that Ad GDNF can sustain increased levels of biosynthesized GDNF in the terminal region of DA neurons for at least 7 weeks and that this GDNF slows the degeneration of DA neurons and prevents the appearance of dopamine dependent motor asymmetries in a rat model of Parkinsons disease (PD). GDNF gene therapy targeted to the striatum, a more surgically accessible site than the SN, may be clinically applicable to humans with PD.

Impaired water maze learning performance without altered dopaminergic function in mice heterozygous for the GDNF mutation.

Exogenous glial cell line‐derived neurotrophic factor (GDNF) exhibits potent survival‐promoting effects on dopaminergic neurons of the nigrostriatal pathway that is implicated in Parkinsons disease and also protects neurons in forebrain ischemia of animal models. However, a role for endogenous GDNF in brain function has not been established. Although mice homozygous for a targeted deletion of the GDNF gene have been generated, these mice die within hours of birth because of deficits in kidney morphogenesis, and, thus, the effect of the absence of GDNF on brain function could not be studied. Herein, we sought to determine whether adult mice, heterozygous for a GDNF mutation on two different genetic backgrounds, demonstrate alterations in the nigrostriatal dopaminergic system or in cognitive function. While both neurochemical and behavioural measures suggested that reduction of GDNF gene expression in the mutant mice does not alter the nigrostriatal dopaminergic system, it led to a significant and selective impairment of performance in the spatial version of the Morris water maze. A standard panel of blood chemistry tests and basic pathological analyses did not reveal alterations in the mutants that could account for the observed performance deficit. These results suggest that endogenous GDNF may not be critical for the development and functioning of the nigrostriatal dopaminergic system but it plays an important role in cognitive abilities.

Genetic Expression Profile of Olfactory Ensheathing Cells Is Distinct From That of Schwann Cells and Astrocytes

Olfactory ensheathing cells (OECs) accompany the axons of olfactory receptor neurons, which regenerate throughout life, from the olfactory mucosa into the olfactory bulb. OECs have shown widely varying efficacy in repairing the injured nervous system. Analysis of the transcriptome of OECs will help in understanding their biology and will provide tools for investigating the mechanisms of their efficacy and interactions with host tissues in lesion models. In this study, we compared the transcriptional profile of cultured OECs with that of Schwann cells (SCs) and astrocytes (ACs), two glial cell types to which OECs have similarities. Two biological replicates of RNA from cultured OECs, SCs, and ACs were hybridized to long oligo rat 5K arrays against a common reference pool of RNA (50% cultured fibroblast RNA and 50% neonatal rat brain RNA). Transcriptional profiles were analyzed by hierarchical clustering, Principal Components Analysis, and the Venn diagram. The three glial cell types had similarly increased or decreased expression of numerous transcripts compared with the reference. However, OECs were distinguishable from both SCs and ACs by a modest number of transcripts, which were significantly enriched or depleted. Furthermore, OECs and SCs were more closely related to each other than to ACs. Expression of selected transcripts not previously characterized in OECs, such as Lyz, Timp2, Gro1 (Cxcl1), Ccl2 (MCP1), Ctgf, and Cebpb, was validated by real‐time reverse transcription‐polymerase chain reaction (RT‐PCR); immunohistochemistry in cultured OECs, SCs, and ACs, and adult tissues was performed to demonstrate their expression at the protein level.

Olfactory ensheathing cells promote collateral axonal branching in the injured adult rat spinal cord

In recent years, injection of olfactory ensheathing cells (ECs) into the spinal cord has been used as an experimental strategy to promote regeneration of injured axons. In this study, we have compared the effects of transplanting encapsulated ECs with those injected directly into the spinal cord. The dorsal columns of adult rats were cut at T(8-9) and rats in experimental groups received either EC-filled porous polymer capsules or culture medium (CM)-filled capsules with ECs injected at the injury site. Control rats were in three groups: (1) uninjured, (2) lesion with transplantation of CM-filled capsules and (3) lesion with transplantation of CM-filled capsules and injections of CM. Three weeks after injury, Fluororuby was injected into the hindlimb motor and somatosensory cortex to label corticospinal neurons. Observations indicated that there were a few regenerating fibres, up to 10, in the EC-treated groups. In rats that received encapsulated ECs, regenerating fibres were present in close association with the capsule. Rats that received EC injections demonstrated a significant increase in the number of collateral branches from the intact ventral corticospinal tract (vCST) compared with the corresponding control, CM-injected group (P=0.003), while a trend for increased collateral branches was observed in rats that received encapsulated ECs (P=0.07).

Adenovirus-Mediated Transgene Expression in Nonhuman Primate Brain

Transgene expression in the brain of St. Kitts green monkey, Cercopithecus aethiops sabeus, was studied following injection of a serotype 5 adenoviral vector deleted in E1 and E3. The vector harbored the transgene for Escherichia coli beta-galactosidase (beta-Gal) with the simian virus 40 (SV40) nuclear localization signal under control of the Rous sarcoma viral (RSV) long terminal repeat. Several titers ranging from 5 x 10(7) to 2 x 10(9) plaque-forming units (PFU) in volumes ranging from 5 to 250 microl were injected into the caudate nuclei of 18 monkeys. Monkeys were treated with dexamethasone for 9 days, beginning the day prior to surgery, and were sacrificed at 1 week or at 1, 2, or 3 months. At 1 week, beta-Gal was expressed in thousands of cells, including both neurons and astrocytes. In addition, some dopaminergic neurons in the substantia nigra expressed transgene, suggesting retrograde transport of the vector. At 1 month 162,000+/-68,000 (SEM) or 65,000+/-29,000 beta-Gal-expressing cells persisted in striatum injected with 6 x 10(8) PFU in 30 microl or 5 x 10(7) PFU in 5 microl, respectively. Transgene expression was also observed in one of two monkeys sacrificed at 2 months and in a single monkey sacrificed at 3 months. No transgene expression was observed at 1 month in striatum injected with a higher titer (2 x 10(9) PFU in 100 microl) or more dilute vector (5 x 10(7) PFU in 30 microl). Staining for the major histocompatibility complex II (MHC II) subtype DR showed intense staining in sites injected with a higher vector titer, in which no transgene persisted at 1 month, whereas low to moderate staining was present in sites with high transgene expression. These observations suggest that there is an optimal range of vector titers for obtaining persistent transgene expression from E1E3-deleted adenovirus in primate brain, above which host responses limit transgene stability.

Bacteria and PAMPs activate nuclear factor κB and Gro production in a subset of olfactory ensheathing cells and astrocytes but not in Schwann cells

The primary olfactory nerves provide uninterrupted conduits for neurotropic pathogens to access the brain from the nasal cavity, yet infection via this route is uncommon. It is conceivable that olfactory ensheathing cells (OECs), which envelope the olfactory nerves along their entire length, provide a degree of immunological protection against such infections. We hypothesized that cultured OECs would be able to mount a biologically significant response to bacteria and pathogen‐associated molecular patterns (PAMPs). The response of OECs to Escherichia coli (E. coli) and various PAMPs was compared to that of Schwann cells (SCs), astrocytes (ACs), and microglia (MG). A subset of OECs displayed nuclear localization of nuclear factor κB), an inflammatory transcription factor, after treatment with E. coli (20% ± 5%), lipopolysacchride (33% ± 9%), and Poly I:C (25% ± 5%), but not with peptidoglycan or CpG oligonucleotides. ACs displayed a similar level of activation to these treatments, and in addition responded to peptidoglycan. The activation of OECs and ACs was enhanced by coculture with MG (56% ± 16% and 85% ± 13%, respectively). In contrast, SCs did not respond to any treatment or to costimulation by MG. Immunostaining for the chemokine Gro demonstrated a functional response that was consistent with NFκB activation. OECs expressed mRNA for Toll‐like receptors (TLRs) 2 and 4, but only TLR4 protein was detected by Western blotting and immunohistochemistry. The results demonstrate that OECs possess the cellular machinery that permits them to respond to certain bacterial ligands, and may have an innate immune function in protecting the CNS against infection.

Homer regulates calcium signalling in growth cone turning

BackgroundHomer proteins are post-synaptic density proteins with known functions in receptor trafficking and calcium homeostasis. While they are key mediators of synaptic plasticity, they are also known to function in axon guidance, albeit by mechanisms that are yet to be elucidated. Homer proteins couple extracellular receptors – such as metabotropic glutamate receptors and the transient receptor potential canonical family of cation channels – to intracellular receptors such as inositol triphosphate and ryanodine receptors on intracellular calcium stores and, therefore, are well placed to regulate calcium dynamics within the neural growth cone. Here we used growth cones from dorsal root ganglia, a well established model in the field of axon guidance, and a growth cone turning assay to examine Homer1 function in axon guidance.ResultsHomer1 knockdown reversed growth cone turning from attraction to repulsion in response to the calcium-dependent guidance cues brain derived neurotrophic factor and netrin-1. Conversely, Homer1 knockdown had no effect on repulsion to the calcium-independent guidance cue Semaphorin-3A. This reversal of attractive turning suggested a requirement for Homer1 in a molecular switch. Pharmacological experiments confirmed that the operational state of a calcium-calmodulin dependent protein kinase II/calcineurin phosphatase molecular switch was dependent on Homer1 expression. Calcium imaging of motile growth cones revealed that Homer1 is required for guidance-cue-induced rise of cytosolic calcium and the attenuation of spontaneous cytosolic calcium transients. Homer1 knockdown-induced calcium transients and turning were inhibited by antagonists of store-operated channels. In addition, immunocytochemistry revealed the close association of Homer1 with the store-operated proteins TRPC1 and STIM1 within dorsal root ganglia growth cones.ConclusionThese experiments provide evidence that Homer1 is an essential component of the calcium signalling repertoire within motile growth cones, regulating guidance-cue-induced calcium release and maintaining basal cytosolic calcium.

Evidence for a novel neurotrophic factor for dopaminergic neurons secreted from mesencephalic glial cell lines

Our previous studies have shown that primary mesencephalic glia secrete factors that promote dopaminergic cell survival and differentiation in vitro. To obtain enough starting material to identify the neurotrophic activity, embryonic day (E)14.5 rat mesencephalic glia were stimulated with acidic fibroblast growth factor to increase number of cells. These cells were replated in the absence of neurons and immortalized by transfection with the SV 40 large T‐antigen. Clonal cell lines were established and characterized for immunoreactivity (IR) to various glial and non‐glial markers. Media conditioned by these cell lines were tested for survival‐promoting effects on dopaminergic neurons in serum‐free cultures of the dissociated E14.5 rat mesencephalon. All cell lines expressed IR for the astrocytic marker, GFAP, the oligodendroglial marker, CNP, and for A2B5, a marker for O‐2A progenitor cells, but were negative for the neuronal marker, microtubule associated protein‐2, and the fibroblast marker, fibronectin. Moreover, treatment of serum‐free cultures of the dissociated E14.5 mesencephalon with glial cell line‐conditioned medium (CM) delayed dopaminergic cell death in a dose‐dependent manner, resulting in a maximal twofold to sixfold increase in the number of surviving tyrosine hydroxylase‐IR neurons at various days in vitro. This increase in dopaminergic cell survival was not mimicked by GDNF, BDNF or NT‐3 within the initial 3 days of cultivation. Moreover, initial biochemical characterization demonstrated that the neurotrophic activity is restricted to the high MW fraction of >50 kD of glial cell line‐CM. Since the apparent MW of this factor exceeds the size of most known growth factors, it may represent a novel dopaminergic neurotrophic factor.