Identification of Stk25 as a genetic modifier of Tau phosphorylation in Dab1-mutant mice.

Hyperphosphorylation of the microtubule binding protein Tau is a feature of a number of neurodegenerative diseases, including Alzheimer's disease. Tau is hyperphosphorylated in the hippocampus of dab1-null mice in a strain-dependent manner; however, it has not been clear if the Tau phosphorylation phenotype is a secondary effect of the morbidity of these mutants. The dab1 gene encodes a docking protein that is required for normal brain lamination and dendritogenesis as part of the Reelin signaling pathway. We show that dab1 gene inactivation after brain development leads to Tau hyperphosphorylation in anatomically normal mice. Genomic regions that regulate the phospho Tau phenotype in dab1 mutants have previously been identified. Using a microarray gene expression comparison between dab1-mutants from the high-phospho Tau expressing and low-phospho Tau expressing strains, we identified Stk25 as a differentially expressed modifier of dab1-mutant phenotypes. Stk25 knockdown reduces Tau phosphorylation in embryonic neurons. Furthermore, Stk25 regulates neuronal polarization and Golgi morphology in an antagonistic manner to Dab1. This work provides insights into the complex regulation of neuronal behavior during brain development and provides insights into the molecular cascades that regulate Tau phosphorylation.

Biochemical and pathological evaluation of long-lived mice with globoid cell leukodystrophy after bone marrow transplantation.

Globoid cell leukodystrophy (GLD) is a disorder of the central and peripheral nervous systems caused by the deficiency of the lysosomal enzyme galactocerebrosidase (GALC). The pathological changes associated with the disease include accumulation of globoid cells and loss of myelin due to production of psychosine, a toxic metabolite responsible for the apoptosis of oligodendrocytes. While most patients present with symptoms before 6 months of age, older patients are also diagnosed. Treatment at this time is limited to hematopoietic stem cell transplantation in asymptomatic and late-onset patients. GLD occurs naturally in several animal species including mice, dogs, and monkeys. In addition, a transgenic (trs) mouse model of GLD was generated in our laboratory. Trs mice develop symptoms slower than twitcher mice and survive an average of 10 days longer. In this study, we evaluated the therapeutic effects of bone marrow transplantation (BMT) using trs mice. BMT prolonged the life of some treated animals to over one year. After BMT, GALC activity reached 15-20% of normal in brain and near normal values in liver and sciatic nerve. In long-lived transplanted animals psychosine levels were normalized in the brain and greatly reduced in the sciatic nerve. Staining of brain sections showed more abundant and better quality myelin and near absence of globoid cells. Electron micrographs of sciatic nerves showed reduced endoneurial edema, increased axon density, and abundant onion bulb structures associated with remyelinating axons. Therefore, BMT can ameliorate many of the biochemical and pathological features of GLD. However, additional therapies may be required to completely correct the features of this disease.

Insulin-like growth factor-1 provides protection against psychosine-induced apoptosis in cultured mouse oligodendrocyte progenitor cells using primarily the PI3K/Akt pathway.

Psychosine (galactosylsphingosine) is a toxic metabolite that accumulates in globoid cell leukodystrophy (GLD) due to the deficiency of galactocerebrosidase (GALC) activity. This results in subsequent programmed cell death of oligodendrocytes and demyelination in human patients and animal models. We investigated the potential role of insulin-like growth factor-1 (IGF-1) in modifying the apoptotic effect of psychosine in cultured mouse oligodendrocyte progenitor cells (OLP-II). We show that psychosine inhibits the phosphorylation of Akt and Erk1/Erk2 (Erk1/2), which are the main anti-apoptotic pathways of the IGF-1 receptor (IGF-1R). Although IGF-1 sustained phosphorylation of both of these pathways, it provided maximum protection to OLP-II cells from psychosine-induced cell death in a PI3K/Akt-dependent manner. The effects of IGF-1 were dose-dependent and resulted in increased IGF-1R autophosphorylation levels. Although relatively high concentrations of IGF-1 also resulted in the activation of the insulin receptor (IR), its effect was more significant on the IGF-1R.

AAV-mediated expression of galactocerebrosidase in brain results in attenuated symptoms and extended life span in murine models of globoid cell leukodystrophy.

Globoid cell leukodystrophy (GLD) or Krabbe disease is a neurodegenerative disorder caused by a deficiency of galactocerebrosidase (GALC) activity. GALC is required for the lysosomal degradation of galactosylceramide, psychosine, and possibly other galactolipids. This process is extremely important during active myelination. In the absence of functional GALC, psychosine accumulates, resulting in the apoptotic death of myelin-producing cells. While most patients are infants who do not survive beyond 2 years of age, some older patients are also diagnosed. Hematopoietic stem cell transplantation has proven to have a positive effect on the course of some patients with late-onset Krabbe disease. Murine models of this disease provide an excellent opportunity to evaluate therapeutic alternatives including gene therapy. In this study we used serotype 1 AAV to express mouse GALC under the control of the human cytomegalovirus promoter. Direct administration of these viral particles into the brains of neonatal mice with GLD resulted in sustained expression of GALC activity, improved myelination, attenuated symptoms, and prolonged life span. While this treatment also resulted in significant pathological improvements, the treated mice died with symptoms similar to those of the untreated mice. Additional initiatives may be required to prevent the onset of disease and reverse the course of the disease in animal models and human patients.

Generation of transgenic mice expressing insulin-like growth factor-1 under the control of the myelin basic protein promoter: increased myelination and potential for studies on the effects of increased IGF-1 on experimentally and genetically induced demyelination.

In order to investigate a role for insulin-like growth factor-1 (IGF-1) in ameliorating the effects of demyelinating events and potentiating remyelination, we have generated transgenic (tg) mice expressing IGF-1 under the control of the myelin basic protein promoter. Heterozygous tg mice expressed the highest levels of IGF-1 in brain during the most active periods of myelination as determined by Western and Northern blotting. A high level of expression was found throughout the lives of the tg mice. There was no increased expression of IGF-1 in other organs. The brains of heterozygous mice were larger than those of normal mice by 2 weeks of age, and they continued to increase in size for several months. Light and electron microscopy showed extensive myelination of axons. Behavioral studies of the older heterozygous mice documented difficulty with balance. This new tg mouse model can be bred to mice that are heterozygous for genetic leukodystrophies to produce eventually mice that are affected with a given leukodystrophy but overexpress IGF-1 during myelination and remyelination. It will be interesting to see if overexpression of IGF-1 can modulate the pathological and clinical features of the inherited leukodystrophies with or without supplemental therapies.

Psychosine-induced apoptosis in a mouse oligodendrocyte progenitor cell line is mediated by caspase activation.

Globoid cell leukodystrophy (GLD) is an inherited neurological disorder caused by the deficiency of galactocerebrosidase (GALC) activity resulting in cell death of oligodendrocytes and subsequent demyelination. The death of oligodendrocytes is accompanied by accumulation of psychosine, which is also a substrate for the GALC enzyme. In this report, we investigated the mechanism of the toxic effect of psychosine in a mouse-derived oligodendrocyte progenitor cell line (OLP-II), a precursor to the cell type most affected in GLD. Psychosine caused cytotoxicity in a dose-dependent manner. Lower concentration of psychosine (5 microM) did not significantly reduce OLP-II cell numbers. However, 50 microM psychosine induced up to 45% cell death. These results were confirmed by the Tunel assay, which is a hallmark for the detection of apoptosis. Moreover, psychosine treatment resulted in the activation/cleavage of initiator caspase-8 and -9, and effector caspase-3. These results support a role for psychosine in OLP-II cell death via an apoptotic mechanism, and suggest the involvement of caspases.