Aj Vincent

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).

Morphological plasticity of olfactory ensheathing cells is regulated by cAMP and endothelin-1

Olfactory ensheathing cells (ECs) are a promising tool for the repair of injury in the adult central nervous system. However, important aspects of the cell biology of ECs remain unclear, such as whether ECs exist as a single population or as two subpopulations with Schwann cell‐like and astrocyte‐like characteristics. The morphologies of these subpopulations are used as defining characteristics, yet ECs are known to be morphologically plastic. To elucidate this apparent inconsistency, we investigated the morphological plasticity of ECs in culture. We defined purified ECs as immunopositive for both p75 neurotrophin receptor and glial fibrillary acidic protein. In MEM D‐valine modification + 10% dialyzed fetal calf serum, 87%–90% of ECs displayed a flat morphology. In three different serum‐free media (N2 medium, neurobasal medium + B27 supplement, and DMEM/F‐12 medium + G5 supplement), 78%–84% of ECs displayed process‐bearing morphology. Ensheathing cells switched reversibly between these morphologies within a day of the serum conditions being changed. Exposure to 1 nM endothelin‐1 in serum‐free medium prevented the switch from flat to process‐bearing morphology, while 1 mM dibutyryl cAMP accelerated this change. The effects of both agents were completely reversible and similar to that reported for astrocytes. Both flat and process‐bearing ECs were immunopositive for brain‐derived neurotrophic factor, nerve growth factor, neurotrophin‐4, and TrkB but not TrkA. Together, these results suggest that ECs exist as a single morphologically plastic population. GLIA 41:393–403, 2003.

The Role of Aβ-Induced Calcium Dysregulation in the Pathogenesis of Alzheimer's Disease

Although many of the biochemical mechanisms which regulate production or clearance of the amyloid-beta protein (Abeta) of Alzheimers disease (AD) are now well understood, the mechanism of Abeta neurotoxicity remains unclear. A number of studies have shown that Abeta can disrupt neuronal Ca(2+) homeostasis by inducing influx of extracellular Ca(2+) into the neuronal cytoplasm. Ca(2+) is known to play an important role in neuronal excitability, synaptic plasticity and neurotoxicity. Therefore, Abeta-induced Ca(2+) dysregulation may contribute to many of the cognitive and neuropathologic features of AD. In vitro studies show that Abeta can increase ion permeability in lipid membranes. This increased permeability is reportedly associated with the formation of artificial ion pores formed from Abeta oligomers. However, a number of other studies show that Abeta can activate endogenous ion channels on the cell surface. There is also increasing evidence that presenilin mutations alter intracellular Ca(2+) stores. It is likely that elucidation of the mechanism by which Abeta and presenilin cause Ca(2+) dysregulation in neurons will help to identify new drug targets for the treatment of AD.

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.

Astrocytes in Alzheimer's disease: emerging roles in calcium dysregulation and synaptic plasticity.

Alzheimers disease (AD) is caused by the accumulation of amyloid-β (Aβ), which induces progressive decline in learning, memory, and other cognitive functions. Aβ is a neurotoxic protein that disrupts calcium signaling in neurons and alters synaptic plasticity. These effects lead to loss of synapses, neural network dysfunction, and inactivation of neuronal signaling. However, the precise mechanism by which Aβ causes neurodegeneration is still not clear, despite decades of intensive research. The role of astrocytes in early cognitive decline is a major component of disease pathology that has been poorly understood. Recent research suggests that astrocytes are not simply passive support cells for neurons, but are active participants in neural information processing in the brain. Aβ can disrupt astrocytic calcium signaling and gliotransmitter release, processes that are vital for astrocyte-neuron communication. Therefore, astrocyte dysfunction may contribute to the earliest neuronal deficits in AD. Here we discuss emerging concepts in glial biology and the implications of astrocyte dysfunction on neurodegeneration in AD.

SPARC is expressed by macroglia and microglia in the developing and mature nervous system

SPARC (secreted protein, acidic and rich in cysteine) is a matricellular protein that is highly expressed during development, tissue remodeling, and repair. SPARC produced by olfactory ensheathing cells (OECs) can promote axon sprouting in vitro and in vivo. Here, we show that in the developing nervous system of the mouse, SPARC is expressed by radial glia, blood vessels, and other pial‐derived structures during embryogenesis and postnatal development. The rostral migratory stream contains SPARC that becomes progressively restricted to the SVZ in adulthood. In the adult CNS, SPARC is enriched in specialized radial glial derivatives (Müller and Bergmann glia), microglia, and brainstem astrocytes. The peripheral glia, Schwann cells, and OECs express SPARC throughout development and in maturity, although it appears to be down‐regulated with maturation. These data suggest that SPARC may be expressed by glia in a spatiotemporal manner consistent with a role in cell migration, neurogenesis, synaptic plasticity, and angiogenesis. Developmental Dynamics 237:1449‐1462, 2008.

SPARC Regulates Microgliosis and Functional Recovery following Cortical Ischemia

Secreted protein acidic rich in cysteine (SPARC) is a matricellular protein that modulates the activity of growth factors, cytokines, and extracellular matrix to play multiple roles in tissue development and repair, such as cellular adhesion, migration, and proliferation. Throughout the CNS, SPARC is highly localized in mature ramified microglia, but its role in microglia—in development or during response to disease or injury—is not understood. In the postnatal brain, immature amoeboid myeloid precursors only induce SPARC expression after they cease proliferation and migration, and transform into mature, ramified resting microglia. SPARC null/CX3CR1-GFP reporter mice reveal that SPARC regulates the distribution and branching of mature microglia, with significant differences between cortical gray and white matter in both controls and SPARC nulls. Following ischemic and excitotoxic lesion, reactive, hypertrophic microglia rapidly downregulate and release SPARC at the lesion, concomitant with reactive, hypertrophic perilesion astrocytes upregulating SPARC. After photothrombotic stroke in the forelimb sensorimotor cortex, SPARC nulls demonstrate enhanced microgliosis in and around the lesion site, which accompanies significantly enhanced functional recovery by 32 d after lesion. Microglia from SPARC nulls also intrinsically proliferate at a greater rate in vitro—an enhanced effect that can be rescued by the addition of exogenous SPARC. SPARC is thus a novel regulator of microglial proliferation and structure, and, in addition to regulating glioma progression, may play an important role in differently regulating the gray and white matter microglial responses to CNS lesion—and modulating behavioral recovery—after injury.

Spinal cord tissue affects ensheathing cell proliferation and apoptosis

This study investigates proliferation and apoptosis of olfactory ensheathing cells in cocultures with spinal cord tissue. Proliferation of ensheathing cells was significantly increased when cocultured with explants from uninjured spinal cord, and spinal cord that had been subjected to chronic contusion or chronic needle stab injury, but not to acute needle stab injury. Proliferation rate was highest in cocultures with chronically stabbed cord tissue. Contaminating (p75NGFR-negative) cells in the cultures showed a significantly higher proliferation rate than ensheathing cells. Apoptosis of ensheathing cells was significantly increased in cocultures with acutely stabbed spinal cord explants compared with chronically contused spinal cord explants. These results suggest that delaying transplantation after spinal cord injury may be beneficial to ensheathing cell survival.

TRPM8 and Nav1.8 sodium channels are required for transthyretin-induced calcium influx in growth cones of small-diameter TrkA-positive sensory neurons

BackgroundFamilial amyloidotic polyneuropathy (FAP) is a peripheral neuropathy caused by the extracellular accumulation and deposition of insoluble transthyretin (TTR) aggregates. However the molecular mechanism that underlies TTR toxicity in peripheral nerves is unclear. Previous studies have suggested that amyloidogenic proteins can aggregate into oligomers which disrupt intracellular calcium homeostasis by increasing the permeability of the plasma membrane to extracellular calcium. The aim of the present study was to examine the effect of TTR on calcium influx in dorsal root ganglion neurons.ResultsLevels of intracellular cytosolic calcium were monitored in dorsal root ganglion (DRG) neurons isolated from embryonic rats using the calcium-sensitive fluorescent indicator Fluo4. An amyloidogenic mutant form of TTR, L55P, induced calcium influx into the growth cones of DRG neurons, whereas wild-type TTR had no significant effect. Atomic force microscopy and dynamic light scattering studies confirmed that the L55P TTR contained oligomeric species of TTR. The effect of L55P TTR was decreased by blockers of voltage-gated calcium channels (VGCC), as well as by blockers of Nav1.8 voltage-gated sodium channels and transient receptor potential M8 (TRPM8) channels. siRNA knockdown of TRPM8 channels using three different TRPM8 siRNAs strongly inhibited calcium influx in DRG growth cones.ConclusionsThese data suggest that activation of TRPM8 channels triggers the activation of Nav1.8 channels which leads to calcium influx through VGCC. We suggest that TTR-induced calcium influx into DRG neurons may contribute to the pathophysiology of FAP. Furthermore, we speculate that similar mechanisms may mediate the toxic effects of other amyloidogenic proteins such as the β-amyloid protein of Alzheimers disease.