Steven G. Matsumoto

Human Neural Stem Cells Induce Functional Myelination in Mice with Severe Dysmyelination

Transplanted banked human neural stem cells produce functional myelin detected by MRI in juvenile mice with severe dysmyelination. Bringing Insulation Up to Code Faulty insulation around household wiring is an electric shock and fire hazard; likewise, defects in the insulation around nerve fibers—the myelin sheath—can have destructive effects. Because of myelin’s crucial roles in promoting the rapid transmission of nerve impulses and in axon integrity, mutations that affect myelin formation in the central nervous system cause severe neurological decline. Uchida et al. and Gupta et al. now investigate the use of neural stem cells—which can differentiate into myelin-producing oligodendrocytes—as a potential treatment for such disorders. Previous work showed that transplantation of human oligodendrocyte progenitors into newborn shiverer (Shi) mice, a hypomyelination model, could prolong survival. In the new work, Uchida et al. transplanted human neural stem cells, which had been expanded and banked, into the brains of newborn and juvenile Shi mice. Whereas the newborn mice were asymptomatic, the juvenile mice were already symptomatic and displayed advanced dysmyelination. These transplanted cells preferentially differentiated into oligodendrocytes that generated myelin, which ensheathed axons and improved nerve conduction in both categories of mice. In an open-label phase 1 study, Gupta et al. then tested the safety and efficacy of such cells in four young boys with a severe, fatal form of Pelizaeus-Merzbacher disease (PMD), a rare X-linked condition in which oligodendrocytes cannot myelinate axons. Human neural stem cells were transplanted directly into the brain; the procedure and transplantation were well tolerated. Magnetic resonance imaging techniques, performed before transplant and five times in the following year, were used to assess myelination. The imaging results were consistent with donor cell–derived myelination in the transplantation region in three of the four patients. These results support further study of potential clinical benefits of neural stem cell transplantation in PMD and other dysmyelination disorders. Shiverer-immunodeficient (Shi-id) mice demonstrate defective myelination in the central nervous system (CNS) and significant ataxia by 2 to 3 weeks of life. Expanded, banked human neural stem cells (HuCNS-SCs) were transplanted into three sites in the brains of neonatal or juvenile Shi-id mice, which were asymptomatic or showed advanced hypomyelination, respectively. In both groups of mice, HuCNS-SCs engrafted and underwent preferential differentiation into oligodendrocytes. These oligodendrocytes generated compact myelin with normalized nodal organization, ultrastructure, and axon conduction velocities. Myelination was equivalent in neonatal and juvenile mice by quantitative histopathology and high-field ex vivo magnetic resonance imaging, which, through fractional anisotropy, revealed CNS myelination 5 to 7 weeks after HuCNS-SC transplantation. Transplanted HuCNS-SCs generated functional myelin in the CNS, even in animals with severe symptomatic hypomyelination, suggesting that this strategy may be useful for treating dysmyelinating diseases.

Digestion products of the PH20 hyaluronidase inhibit remyelination

Oligodendrocyte progenitor cells (OPCs) recruited to demyelinating lesions often fail to mature into oligodendrocytes (OLs) that remyelinate spared axons. The glycosaminoglycan hyaluronan (HA) accumulates in demyelinating lesions and has been implicated in the failure of OPC maturation and remyelination. We tested the hypothesis that OPCs in demyelinating lesions express a specific hyaluronidase, and that digestion products of this enzyme inhibit OPC maturation.

Sensory deprivation during development decreases the responsiveness of cricket giant interneurones.

1. The effect sensory deprivation, early in development, has on the adult response properties of identified neurones was studied in the abdominal nervous system of the cricket Acheta domesticus.

Hyaluronan Anchored to Activated CD44 on Central Nervous System Vascular Endothelial Cells Promotes Lymphocyte Extravasation in Experimental Autoimmune Encephalomyelitis

Background: Multiple sclerosis (MS) is a demyelinating disease involving lymphocyte infiltration into the central nervous system (CNS). Results: The glycosaminoglycan hyaluronan (HA), anchored to brain blood vessels via the CD44 receptor, facilitates lymphocyte binding to vessels and CNS infiltration. Conclusion: HA-CD44 interactions on brain endothelial cells facilitate the initiation of inflammatory demyelinating disease. Significance: Findings elucidate mechanisms promoting lymphocyte rolling in inflammatory CNS diseases. The extravasation of lymphocytes across central nervous system (CNS) vascular endothelium is a key step in inflammatory demyelinating diseases including multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). The glycosaminoglycan hyaluronan (HA) and its receptor, CD44, have been implicated in this process but their precise roles are unclear. We find that CD44−/− mice have a delayed onset of EAE compared with wild type animals. Using an in vitro lymphocyte rolling assay, we find that fewer slow rolling (<1 μm/s) wild type (WT) activated lymphocytes interact with CD44−/− brain vascular endothelial cells (ECs) than with WT ECs. We also find that CD44−/− ECs fail to anchor HA to their surfaces, and that slow rolling lymphocyte interactions with WT ECs are inhibited when the ECs are treated with a pegylated form of the PH20 hyaluronidase (PEG-PH20). Subcutaneous injection of PEG-PH20 delays the onset of EAE symptoms by ∼1 day and transiently ameliorates symptoms for 2 days following disease onset. These improved symptoms correspond histologically to degradation of HA in the lumen of CNS blood vessels, decreased demyelination, and impaired CD4+ T-cell extravasation. Collectively these data suggest that HA tethered to CD44 on CNS ECs is critical for the extravasation of activated T cells into the CNS providing new insight into the mechanisms promoting inflammatory demyelinating disease.

Transmitter status in cultured sympathetic principal neurons: plasticity, graded expression and diversity.

Publisher Summary This chapter summarizes the recent investigations of the transmitter status of sympathetic principal neurons derived from neonatal or adult rats and grown singly in microcultures with cardiac cells. The work began out of an interest in the status of individual neonatal sympathetic neurons during a transition from an initial (at least) adrenergic state to a predominantly cholinergic state, under the influence of non-neuronal cells. Under the influence of nerve growth factor, neurites grow progressively over the microculture but not beyond its borders. Many microcultures survive for 1–3 months; after such periods, the density of neurites over the myocytes is often greater than that of the normal innervation of sympathetic target tissues in vivo . The classical view of transmitter status in adult mammalian sympathetic principal neurons is that two transmitters—norepinephrine and acetylcholine—are expressed. Each neuron secretes only one transmitter (monofunction), and that transmitter is expressed approximately full-on; once the appropriate transmitter is adopted, the neuron does not change status.

Calcium transient activity in cultured murine neural crest cells is regulated at the IP3 receptor

In a previous study we have shown that cultured neural crest cells exhibit spontaneous calcium transients and that these events are required for neurogenesis. In this study, we examine the mechanism that generates these calcium transients. Extracellular Ca(2+) modulates calcium transient activity. Lanthanum (La(3+)), a general calcium channel antagonist and zero extracellular Ca(2+), reduces the percentage of cells exhibiting calcium transients (26.2 and 40. 5%, respectively) and decreases calcium spiking frequency (4.5 to 1. 0 and 2.5 to 1.0 spikes/30 min, respectively). Intracellular calcium stores also contribute to the generation of calcium transients. Depleting the calcium stores of the endoplasmic reticulum (ER) reduces the percentage of active cells (15.7%) and calcium spiking frequency (2.8 to 1.5 spikes/30 min). Ryanodine (100 microM), which blocks calcium release regulated by the ryanodine receptor (RyR), had no effect on calcium transient activity. Blocking inositol 1,4, 5-triphosphate receptor (IP(3)R)-dependent calcium release, with elevated extracellular Mg(2+) (20 mM), abolished calcium transient activity. Mg(2+) did not block caffeine-sensitive calcium release (RyR-dependent) or voltage dependent calcium channels. Mg(2+) also suppressed thimerosal-induced calcium oscillations (IP(3)R-dependent). Small increases in the intracellular calcium concentration ([Ca(2+)](i)), increases the percentage of active cells and the calcium spiking frequency, while larger increases in [Ca(2+)](i) block the transients. Reducing intracellular IP(3) levels reduces the percentage of active cells and the calcium spiking frequency. We conclude that the mechanism for generating spontaneous calcium transients in cultured neural crest cells fits the model for IP(3)R-dependent calcium excitability of the ER.

Hyaluronan Synthesis, Catabolism, and Signaling in Neurodegenerative Diseases

The glycosaminoglycan hyaluronan (HA), a component of the extracellular matrix, has been implicated in regulating neural differentiation, survival, proliferation, migration, and cell signaling in the mammalian central nervous system (CNS). HA is found throughout the CNS as a constituent of proteoglycans, especially within perineuronal nets that have been implicated in regulating neuronal activity. HA is also found in the white matter where it is diffusely distributed around astrocytes and oligodendrocytes. Insults to the CNS lead to long-term elevation of HA within damaged tissues, which is linked at least in part to increased transcription of HA synthases. HA accumulation is often accompanied by elevated expression of at least some transmembrane HA receptors including CD44. Hyaluronidases that digest high molecular weight HA into smaller fragments are also elevated following CNS insults and can generate HA digestion products that have unique biological activities. A number of studies, for example, suggest that both the removal of high molecular weight HA and the accumulation of hyaluronidase-generated HA digestion products can impact CNS injuries through mechanisms that include the regulation of progenitor cell differentiation and proliferation. These studies, reviewed here, suggest that targeting HA synthesis, catabolism, and signaling are all potential strategies to promote CNS repair.

NEURONAL DIFFERENTIATION IN CULTURES OF MURINE NEURAL CREST. I: NEUROTRANSMITTER EXPRESSION

This study examines the properties of neurons differentiating in cultures of mammalian neural crest cells. The neurons fall into two categories: (1) a population of early differentiating (ED) neurons generated from precursors that are postmitotic at the time of plating; and (2) a late differentiating (LD) population of neurons arising from dividing precursor cells. The ED population of neurons survive for only 2-3 days while the LD neurons survive for many weeks. Both groups of neurons express the neuronal marker, neurofilament, as well as adrenergic and cholinergic characteristics. The latter two traits are evident as immunoreactivity for tyrosine hydroxylase (TH)/dopamine-beta-hydroxylase (D beta H) and choline acetyltransferase (ChAT), respectively. The LD neurons also contain immunoreactivity for a number of neuropeptides including, substance P (SP), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), calcitonin gene related polypeptide (CGRP), and somatostatin (SOM). Immunoreactivity for SP, CGRP, and VIP are found in virtually all of the LD neurons while SOM and NPY are found in a smaller percentage of the neurons.

Neurotransmitter properties of guinea-pig sympathetic neurons grown in dissociated cell culture—II. Fetal and embryonic neurons: Regulation of neuropeptide Y expression

We report here the neurotransmitter characteristics of neurons cultured from the same ganglia of fetal and embryonic guinea-pigs. Both the celiac ganglion and the superior mesenteric ganglion were examined. In a previous paper we described the neurotransmitter properties of adult guinea-pig prevertebral sympathetic neurons grown in dissociated cell culture, including the expression by these cells of immunoreactivity for tyrosine hydroxylase, neuropeptide Y and somatostatin. Tyrosine hydroxylase immunoreactivity was ubiquitously expressed in all fetal embryonic cultures, as was the case for adult neurons. Fetal-derived celiac and superior mesenteric gangli neurons displayed neuropeptide Y and somatostatin immunoreactivity in the same percentage of neurons as in adult cultures but at markedly lower levels. Embryonic neurons also expressed somatostatin immunoreactivity in roughly the same proportion of neurons as in adult and fetal cultures; however, the expression of neuropeptide Y immunoreactivity in both celiac and superior mesenteric gangli cultures was significantly different. Specifically, neuropeptide Y immunoreactivity in embryonic celiac cultures was greatly reduced in both the number of positive-labeled neurons and the amount of immunoreactive product, while neuropeptide Y immunoreactivity in embryonic superior mesenteric gangli cultures was markedly increased compared to their adult and fetal counterparts. The expression of neuropeptide Y immunoreactivity in celiac neurons was found to be specifically elevated by culturing the neurons in medium conditioned by disassociated vascular cells, this treatment having no effect on tyrosine hydroxylase or somatostatin immunoreactivity. Heart cell-conditioned medium did not effect neuropeptide Y or somatostatin immunoreactivity, although it did result in a significant reduction of tyrosine hydroxylase immunoreactivity and an increase in 5-hydroxytryptamine immunoreactivity. We conclude that the expression of neuropeptide Y immunoreactivity develops independently in cultures of adult and near-term fetuses but that embryonic neurons require interactions with target cells to express this phenotype. Neuropeptide Y immunoreactivity can be induced in embryonic sympathetic neurons by a target-derived factor(s).

Hyaluronan oligosaccharides perturb lymphocyte slow rolling on brain vascular endothelial cells: Implications for inflammatory demyelinating disease

Inflammatory demyelinating diseases like multiple sclerosis are characterized by mononuclear cell infiltration into the central nervous system. The glycosaminoglycan hyaluronan and its receptor, CD44, are implicated in the initiation and progression of a mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). Digestion of hyaluronan tethered to brain vascular endothelial cells by a hyaluronidase blocks the slow rolling of lymphocytes along activated brain vascular endothelial cells and delays the onset of EAE. These effects could be due to the elimination of hyaluronan or the generation of hyaluronan digestion products that influence lymphocytes or endothelial cells. Here, we found that hyaluronan dodecasaccharides impaired activated lymphocyte slow rolling on brain vascular endothelial cells when applied to lymphocytes but not to the endothelial cells. The effects of hyaluronan dodecasaccharides on lymphocyte rolling were independent of CD44 and a receptor for degraded hyaluronan, Toll-like receptor-4. Subcutaneous injection of hyaluronan dodecasaccharides or tetrasaccharides delayed the onset of EAE in a manner similar to subcutaneous injection of hyaluronidase. Hyaluronan oligosaccharides can therefore act directly on lymphocytes to modulate the onset of inflammatory demyelinating disease.