Autoimmune Voltage-Gated Potassium Channelopathy Presenting With Catecholamine Excess.

BACKGROUND: Autoimmune voltage-gated potassium channelopathies have been associated with a range of neurological presenting symptoms, including central, peripheral, and autonomic dysfunction. PATIENT DESCRIPTION: We describe a 12-year-old boy who presented with nine months of pain, anxiety, and 30-pound weight loss. He was admitted for failure to thrive, then noted to be persistently hypertensive and tachycardic. Plasma metanephrines and urine metanephrines and catecholamines were elevated. Extensive investigation for causes of elevated catecholamines, such as hyperthyroidism or catecholamine-secreting tumor, was negative. A paraneoplastic panel was positive for voltage-gated potassium channel antibodies. Treatment with intravenous immunoglobulin and pulse methylprednisolone led to complete resolution of symptoms, weight gain, and normalization of vital signs and plasma metanephrines. CONCLUSION: Voltage-gated potassium channel antibodies should be considered as part of the differential in patients presenting with elevated metanephrine and catecholamine secretion.

Cellular fusion for gene delivery to SCA1 affected Purkinje neurons.

Cerebellar Purkinje neurons (PNs) possess a well characterized propensity to fuse with bone marrow-derived cells (BMDCs), producing heterokaryons with Purkinje cell identities. This offers the potential to rescue/repair at risk or degenerating PNs in the inherited ataxias, including Spinocerebellar Ataxia 1 (SCA1), by introducing therapeutic factors through BMDCs to potentially halt or reverse disease progression. In this study, we combined gene therapy and a stem cell-based treatment to attempt repair of at-risk PNs through cell-cell fusion in a Sca1(154Q/2Q) knock-in mouse model. BMDCs enriched for the hematopoietic stem cell (HSC) population were genetically modified using adeno-associated viral vector 7 (AAV7) to carry SCA1 modifier genes and transplanted into irradiated Sca1(154Q/2Q) mice. Binucleated Purkinje heterokaryons with sex-mismatched donor Y chromosomes were detected and successfully expressed the modifier genes in vivo. Potential effects of the new genome within Purkinje heterokaryons were evaluated using nuclear inclusions (NIs) as a biological marker to reflect possible modifications of the SCA1 disease process. An overall decrease in number of NIs and an increase in the number of surviving PNs were observed in treated Sca1(154Q/2Q). Furthermore, Bergmann glia were found to have fusogenic potential with the donor population and reveal another potential route of therapeutic entry into at-risk cells of the SCA1 cerebellum. This study presents a first step towards a proof-of-principle that combines somatic cellular fusion events with a neuroprotective gene therapy approach for providing potential neuronal protection/repair in a variety of neurodegenerative disorders.

Role of individual and developmental differences in voluntary cocaine intake in rats.

RATIONALE: Early-onset drug taking is associated with increased likelihood of addiction, but it is unclear whether early onset is causal in development of addiction. Many other factors are associated with increased risk of addiction and also promote early intake. Here, a rodent model is used to explore the causality of early onset in development of self-administration and addiction-like behavior and to examine factors that promote self-administration. METHODS: We used cocaine self-administration to examine drug taking and addiction-like behavior in adolescent and adult rats a priori characterized for their locomotor responses to novelty and cocaine and behavior in the light-dark task. RESULTS: Adolescent animals initially sought more cocaine than adults. However, as the adolescents matured, their intake fell and they did not differ from adults in terms of unreinforced lever-pressing, extinction or reinstatement behavior. For both age groups, self-administration was positively correlated with the locomotor response to novelty, the locomotor response to cocaine, and with time in light in the light-dark task. The rats that were insensitive to cocaine's locomotor effects and that spent the least time in light in the light-dark task sought the least cocaine, appearing to be "protected" from the reinforcing effects of cocaine. There was no difference between the two age groups in appearance of this "protected" phenotype. CONCLUSIONS: These results suggest that early onset of drug taking may promote increased use, but does not promote progression to addiction-like behavior. Furthermore, protective factors, such as innate anxiety and insensitivity to cocaine's pharmacological effects, function across developmental stages.

Transplantation of embryonic and adult neural stem cells in the granuloprival cerebellum of the weaver mutant mouse.

Numerous studies have explored the potential of different stem and progenitor cells to replace at-risk neuronal populations in a variety of neurodegenerative disease models. This study presents data from a side-by-side approach of engrafting two different stem/progenitor cell populations within the postnatal cerebellum of the weaver neurological mutant mouse--cerebellar-derived multipotent astrocytic stem cells and embryonic stem cell-derived neural precursors--for comparative analysis. We show here that both donor populations survive, migrate, and appear to initiate differentiation into neurons within the granuloprival host environment. Neither of these disparate stem/progenitor cell populations adopted significant region-specific identities, despite earlier studies that suggested the potential of these cells to respond to in vivo cues when placed in a permissive/instructive milieu. However, data presented here suggest that molecular and cellular deficits present within weaver homozygous or heterozygous brains may promote a slightly more positive donor cell response toward acquisition of a neuronal phenotype. Hence, it is likely that a fine balance exists between a compromised host environment that is amenable to cell replacement and that of a degenerating cellular milieu where it is perhaps too deleterious to support extensive neuronal differentiation and functional cellular integration. These findings join a growing list of studies that show successful cell replacement depends largely on the interplay between the potentiality of the donor cells and the specific pathological conditions of the recipient environment, and that emergent therapies for neurological disorders involving the use of neural stem cells still require refinement.

Transplantation of neural stem/progenitor cells into developing and adult CNS.

Neural transplantation has been a long-standing goal for the treatment of neurological injury and disease. The recent discovery of persistent pools of neural stem cells within the adult mammalian brain has re-ignited interest in transplant therapeutics. Since neural stem cells are self-renewing, it may be possible to culture and expand neural stem cells and their progenitor cell progeny to sufficient numbers for use in autologous, self-repair strategies. Such approaches will require optimized cultivation protocols, as well as extensive testing of candidate donor cells to assess their capacity for engraftment, survival, and integration. In this chapter, we describe the transplantation of neural stem/progenitor cells-cultivated as either neurospheres or neurogenic astrocyte monolayers-into the persistently neurogenic olfactory bulb system of the adult mouse forebrain, and into the cerebellum of neonatal mutant mice.

Fusion of neural stem cells in culture.

An important issue in stem cell biology relates to mechanisms of cellular plasticity. Specifically, could any observed multipotency of, e.g., adult stem cells arise from true transdifferentiation or as a result of cell-cell fusion? We studied this issue using a culture paradigm of astrocyte monolayers and multipotent neurospheres generated from neonatal cerebellar cortex and the subventricular zone (SVZ). Based on fluorescence in situ hybridization (FISH), cells from these cultures were found to contain an abnormal number of sex chromosomes, suggesting that cellular fusion is a common in vitro occurrence. A Cre/lox recombination method was also exploited to further confirm the evidence of fusion. Next, we assessed the potential of fusogenic microglial involvement by combining CD11b immunolabeling with FISH sex chromosome analysis. Differentiating neurospheres were also studied from the PU.1 knockout mouse that lacks cells of myeloid origin, presumed to be a source of central nervous system microglia. Very few cells immunopositive for the microglial marker CD11b were found to be aneuploid, and there was no difference in fusion frequency between PU.1+/+ and PU.1-/- neurospheres. These results, together, suggest that stem and/or progenitor cells that generate neurons and glia in culture possess the ability to generate fused polyploidal cells, but microglial participation is not a requirement for fusion to occur. In addition to caution that should be exerted during the interpretation of in vitro neural cell plasticity, the data also suggest that novel therapeutic treatments could be designed that exploit cellular fusion in rescue paradigms for degenerating neuronal populations.

Neuron-to-astrocyte transition: phenotypic fluidity and the formation of hybrid asterons in differentiating neurospheres.

To the extent that their fate choice and differentiation processes can be understood and manipulated, neural stem cells represent a promising therapeutic tool for a variety of neuropathologies. We have previously shown that mature astrocytes possess neural stem cell attributes, and can give rise to neurons through the formation of multipotent neurosphere clones. Here we show that relatively mature neurons generated from neurospheres derived from postnatal subependymal zone or cerebellar cortex undergo a phenotypic transformation into astrocytes that coincides with the appearance of a nonfused, hybrid cell type that shares the morphology, antigenicity, and physiology of both neurons and astrocytes. We refer to this astrocyte/neuron hybrid as an "asteron," and hypothesize that it represents an intermediate step in the trans- or dedifferentiation of neurons into astrocytes. The present finding suggests that seemingly terminally differentiated neural cells may in fact represent points along a bidirectionally fluid continuum of differentiation, with intermediate points represented by "hybrid" cells coexpressing phenotypic markers of more than one lineage.

Capillary sieving electrophoresis-micellar electrokinetic chromatography fully automated two-dimensional capillary electrophoresis analysis of Deinococcus radiodurans protein homogenate.

We report the one- and two-dimensional (1-D and 2-D) capillary electrophoresis separation of Deinococcus radiodurans protein homogenate. Proteins are labeled with the fluorogenic reagent 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ), which reacts with lysine residues and creates a highly fluorescent product. Detection is by laser-induced fluorescence. 1-D capillary sieving electrophoresis (CSE) produces over 150,000 plates and micellar electrokinetic capillary chromatography (MEKC) produces over 900,000 plates for components in a D. radiodurans protein homogenate. In a 2-D separation, proteins are first separated by CSE. Fractions are repetitively transferred to a second capillary for further separation based on MEKC. The 2-D separation has a approximately 550 spot capacity. Over 150 components are partially resolved from the homogenate. Resolution is limited in the first dimension by diffusion of proteins during the long separation period and in the second dimension by the combination of a long fraction-transfer time and short separation period.