Nancy R. Cox

Isolation and characterization of multipotential mesenchymal stem cells from feline bone marrow

OBJECTIVE Although several types of stem cells have been isolated from rodent and human tissues, very few data exist on stem cell isolation from nonrodent animals, which seriously limits the advancement of stem cell biology and its ultimate translation to human clinical applications. Domestic cats are used frequently in biomedical research and are the preferred species for studies of normal physiology and disease, particularly in neuroscience. Therefore, the objective of this study was to characterize mesenchymal stem cells (MSC) from feline bone marrow for use in research on the application of stem cells to human health problems for which cats are the preferred model. METHODS Mesenchymal stem cells from feline bone marrow were isolated by standard methodology developed for other species and characterized according to morphology, growth traits, cell-surface antigen profile, and differentiation repertoire in vitro. RESULTS Feline mesenchymal stem cells exhibit a fibroblast-like morphology with bipolar or polygonal cell bodies and possess a cell-surface antigen profile similar to their rodent and human counterparts. Feline MSC exist at a frequency of 1 in 3.8 x 10(5) bone marrow mononuclear cells and are capable of differentiation to adipocytic, osteocytic, and neuronal phenotypes when exposed to appropriate induction media. CONCLUSIONS Mesenchymal stem cells isolated from feline bone marrow possess several traits typical of MSC from other species. Characterization of feline mesenchymal stem cells will facilitate future studies of stem cell biology and therapeutics for which the domestic cat is an indispensable model.

Essential Role of Sequestosome 1/p62 in Regulating Accumulation of Lys63-ubiquitinated Proteins

Sequestosome 1 (SQSTM1)/p62 is an interacting partner of the atypical protein kinase C ζ/ι and serves as a scaffold for cell signaling and ubiquitin binding, which is critical for several cell functions in vivo such as osteoclastogenesis, adipogenesis, and T cell activation. Here we report that in neurons of p62–/– mouse brain there is a detectable increase in ubiquitin staining paralleled by accumulation of insoluble ubiquitinated proteins. The absolute amount of each ubiquitin chain linkage measured by quantitative mass spectrometry demonstrated hyperaccumulation of Lys63 chains in the insoluble fraction recovered from the brain of p62–/– mice, which correlated with increased levels of Lys63-ubiquitinated TrkA receptor. The increase in Lys63 chains was attributed in part to diminished activity of the TRAF6-interacting the Lys63-deubiquitinating enzyme (DUB), cylindromatosis tumor suppressor (CYLD). The interaction of CYLD with TRAF6 was dependent upon p62, thus defining a mechanism that accounts for decreased activity of CYLD in the absence of p62. These findings reveal that p62 serves as an adapter for the formation of this complex, thereby regulating the DUB activity of CYLD by TRAF6 interaction. Thus, p62 has a bifunctional role in regulation of an E3 ubiquitin-protein ligase, TRAF6, and a DUB, CYLD, to balance the turnover of Lys63-polyubiquitinated proteins such as TrkA.

Genetic inactivation of p62 leads to accumulation of hyperphosphorylated tau and neurodegeneration.

The signaling adapter p62 plays a coordinating role in mediating phosphorylation and ubiquitin‐dependent trafficking of interacting proteins. However, there is little known about the physiologic role of this protein in brain. Here, we report age‐dependent constitutive activation of glycogen synthase kinase 3β, protein kinase B, mitogen‐activated protein kinase, and c‐Jun‐N‐terminal kinase in adult p62−/− mice resulting in hyperphosphorylated tau, neurofibrillary tangles, and neurodegeneration. Biochemical fractionation of p62−/− brain led to recovery of aggregated K63‐ubiquitinated tau. Loss of p62 was manifested by increased anxiety, depression, loss of working memory, and reduced serum brain‐derived neurotrophic factor levels. Our findings reveal a novel role for p62 as a chaperone that regulates tau solubility thereby preventing tau aggregation. This study provides a clear demonstration of an Alzheimer‐like phenotype in a mouse model in the absence of expression of human genes carrying mutations in amyloid‐beta protein precursor, presenilin, or tau. Thus, these findings provide new insight into manifestation of sporadic Alzheimer disease and the impact of obesity.

Neural stem/progenitor cells modulate immune responses by suppressing T lymphocytes with nitric oxide and prostaglandin E2

We and others have reported that neural stem/progenitor cells (NSCs) may exert direct anti-inflammatory activity. This action has been attributed, in part, to T-cell suppression. However, how T-cells become suppressed by NSCs remains unresolved. In this study, we explored one of these mechanisms and challenged some previously advanced hypotheses regarding underlying NSC-mediated T-cell suppression. We employed an easily observable and manipulatable system in which activated and non-activated T-cells were co-cultured with a stable well-characterized clone of lacZ-expressing murine NSCs. As in previous reports, NSCs were found to inhibit T-cell proliferation. However, this inhibition by NSCs was not due to suppression of T cell activation or induction of apoptosis of T cells during the early activation stage. High levels of nitric oxide (NO) and prostaglandin E2 (PGE2) were induced in the T cells when co-cultured with NSCs. In addition, inducible NOS (iNOS) and microsomal type 1 PGES (mPGES-1) were readily detected in NSCs in co-culture with T-cells, but not at all in NSCs cultured alone or in activated T cells cultured with or without NSCs. This finding suggested that activated T cells induced NO and PGE2 production in the NSCs. Furthermore, T-cell proliferation inhibited by co-culture with the NSCs was significantly restored by inhibitors of NO and PGE2 production. Therefore, NSCs appear to suppress T-cells, at least in part, by NO and PGE2 production which, in turn, would account for the well-documented reduction of central nervous system immunopathology by transplanted NSCs.

Therapeutic response in feline sandhoff disease despite immunity to intracranial gene therapy.

Salutary responses to adeno-associated viral (AAV) gene therapy have been reported in the mouse model of Sandhoff disease (SD), a neurodegenerative lysosomal storage disease caused by deficiency of β-N-acetylhexosaminidase (Hex). While untreated mice reach the humane endpoint by 4.1 months of age, mice treated by a single intracranial injection of vectors expressing human hexosaminidase may live a normal life span of 2 years. When treated with the same therapeutic vectors used in mice, two cats with SD lived to 7.0 and 8.2 months of age, compared with an untreated life span of 4.5 ± 0.5 months (n = 11). Because a pronounced humoral immune response to both the AAV1 vectors and human hexosaminidase was documented, feline cDNAs for the hexosaminidase α- and β-subunits were cloned into AAVrh8 vectors. Cats treated with vectors expressing feline hexosaminidase produced enzymatic activity >75-fold normal at the brain injection site with little evidence of an immune infiltrate. Affected cats treated with feline-specific vectors by bilateral injection of the thalamus lived to 10.4 ± 3.7 months of age (n = 3), or 2.3 times as long as untreated cats. These studies support the therapeutic potential of AAV vectors for SD and underscore the importance of species-specific cDNAs for translational research.

Comparative Analysis of Brain Lipids in Mice, Cats, and Humans with Sandhoff Disease

Sandhoff disease (SD) is a glycosphingolipid (GSL) storage disease that arises from an autosomal recessive mutation in the gene for the β-subunit of β-Hexosaminidase A (Hexb gene), which catabolizes ganglioside GM2 within lysosomes. Accumulation of GM2 and asialo-GM2 (GA2) occurs primarily in the CNS, leading to neurodegeneration and brain dysfunction. We analyzed the total lipids in the brains of SD mice, cats, and humans. GM2 and GA2 were mostly undetectable in the normal mouse, cat, and human brain. The lipid abnormalities in the SD cat brain were generally intermediate to those observed in the SD mouse and the SD human brains. GM2 comprised 38, 67, and 87% of the total brain ganglioside distribution in the SD mice, cats, and humans, respectively. The ratio of GA2–GM2 was 0.93, 0.13, and 0.27 in the SD mice, cats, and humans, respectively, suggesting that the relative storage of GA2 is greater in the SD mouse than in the SD cat or human. Finally, the myelin-enriched lipids, cerebrosides and sulfatides, were significantly lower in the SD brains than in the control brains. This study is the first comparative analysis of brain lipids in mice, cats, and humans with SD and will be important for designing therapies for Sandhoff disease patients.

Concomitant brainstem axonal dystrophy and necrotizing myopathy in vitamin E-deficient rats

The purpose of this study was to simultaneously evaluate in rats the effects of vitamin E depletion on tissue alpha-tocopherol (alpha-T) concentrations, electrophysiologic measurements and histopathology. Rats (21-day-old male Wistar) were fed either vitamin E-deficient or supplemented (control) diets (n = 6/group) for 10, 16, and 61 weeks. At these times, electrophysiologic tests (electromyography, spinal and somatosensory evoked potentials, and motor nerve conduction velocity) were performed, the rats were killed and alpha-T concentrations of adipose tissue, sciatic nerve, and cervical and lumbar spinal cord were measured along with histopathologic evaluation of skeletal muscles and the nervous system. By 61 weeks, depletion of alpha-T from adipose tissue and peripheral nerve was more severe (< 1% of controls) than from cervical and lumbar spinal cord (15 and 8% of controls, respectively). Electrophysiologic tests were normal at all times. Histopathologic evaluation at 61 weeks revealed normal peripheral nerve structure, but necrosis of type 1 muscle fibers and increased numbers of spheroids in the gracile and cuneate nuclei. Our results confirm that low alpha-T concentrations in tissues precede histologic changes in peripheral nerves and skeletal muscle. Furthermore, pathologic changes associated with vitamin E deficiency occur independently in muscle and nervous tissue of rats.

Targeting peptides for microglia identified via phage display

Screening with a 7-mer phage display peptide library, a panel of cell-targeting peptides for the murine microglial cell line, EOC 20, was recognized. A number of similar, but not identical, sets of sequences representing more than 75% of all the cell line-binding clones were identified. Comparative analysis indicated that motif S/(T) F T/(X) Y W is present in the vast majority of the binding sequences. The selectivity and specificity of the dominant peptide sequence identified for microglia was confirmed using both phage displaying the peptide and the synthetic peptide alone.

α-tocopherol concentrations of the nervous system and selected tissues of adult dogs fed three levels of vitamin E

The effects of dietary vitamin E levels on tissue α-tocopherol (α−T) concentrations in different parts of the nervous system are largely unknown. Therefore, we measured the α−T contents of nervous and other tissues obtained from beagle dogs fed for two years a vitamin E-deficient diet (−E, 0.05±0.02 mg vitamin E/kg diet, n=2), a vitamin E-supplemented diet (+E, 114±14 mg/kg, n=2), or a standard chow diet (En, 74±6 mg/kg, n=3). Brain regions and spinal cords of +E dogs contained about double the α−T concentrations of En dogs, and about 10-fold those of −E dogs. The various brain regions of −E dogs, compared with En dogs, retained 12–18% of the α−T concentrations, with the exception of the caudal colliculus, which retained 48%. Peripheral nerve α−T concentrations in +E dogs (67 ng/mg wet weight) were nearly 5-fold higher than in En dogs (13.4±5.9 ng/mg) and 80-fold higher than in −E dogs (0.8 ng/mg). Within each dietary group, the lowest α−T concentrations in the central nervous system (CNS) were in the spinal cord. Peripheral nerves were the most susceptible to vitamin E repletion or depletion: in +E dogs, nerves contained higher concentrations of α−T than most brain regions; in En dogs, they contained similar concentrations; but in −E dogs, they contained less α−T than most brain regions. Muscles and other tissues of −E dogs retained from 1 to 10% of En values. The studies demonstrate that the CNS conserved α−T compared to peripheral nerves and nonnervous tissues in adult dogs, but contained lower absolute concentrations of α−T compare with most other tissues.

Sustained Normalization of Neurological Disease after Intracranial Gene Therapy in a Feline Model

In a feline model of lysosomal storage disease, intracranial gene therapy achieved therapeutic efficacy in the CNS and increased long-term survival. Gene Therapy for a Lysosomal Storage Disease GM1 gangliosidosis results from defects in the lysosomal enzyme β-galactosidase (β-gal) and subsequent accumulation of GM1 ganglioside, which causes neurodegeneration and premature death. Although no effective treatment exists, encouraging gene therapy data from the GM1 mouse model warranted an evaluation of the feasibility for human clinical application in a large animal model. In a new study, McCurdy et al. injected an adeno-associated viral vector encoding feline β-gal bilaterally into two brain targets (thalamus and deep cerebellar nuclei) of cats with GM1 gangliosidosis. Sixteen weeks after injection, β-gal activity and GM1 storage were normalized throughout the central nervous system of the animals, with accompanying increases in enzyme activity in cerebrospinal fluid and liver. In long-term studies, the mean survival of 12 treated cats with GM1 gangliosidosis was >38 months, compared to 8 months for untreated cats. A minority of cats that progressed to the humane endpoint had low β-gal activity in the spinal cord, yet still lived >2.5 times longer than untreated animals. Most of the treated GM1 cats demonstrated subtle or no gait abnormalities, and magnetic resonance imaging showed normalization of brain architecture up to at least 32 months of age. Long-term correction of the disease phenotype in cats with GM1 gangliosidosis suggests that gene therapy may be useful for treating the human disorder. Progressive debilitating neurological defects characterize feline GM1 gangliosidosis, a lysosomal storage disease caused by deficiency of lysosomal β-galactosidase. No effective therapy exists for affected children, who often die before age 5 years. An adeno-associated viral vector carrying the therapeutic gene was injected bilaterally into two brain targets (thalamus and deep cerebellar nuclei) of a feline model of GM1 gangliosidosis. Gene therapy normalized β-galactosidase activity and storage throughout the brain and spinal cord. The mean survival of 12 treated GM1 animals was >38 months, compared to 8 months for untreated animals. Seven of the eight treated animals remaining alive demonstrated normalization of disease, with abrogation of many symptoms including gait deficits and postural imbalance. Sustained correction of the GM1 gangliosidosis disease phenotype after limited intracranial targeting by gene therapy in a large animal model suggests that this approach may be useful for treating the human version of this lysosomal storage disorder.