Inositol hexakisphosphate kinase 2 promotes cell death of anterior horn cells in the spinal cord of patients with amyotrophic lateral sclerosis.

We have previously reported that inositol hexakisphosphate kinase (InsP6K)2 mediates cell death. InsP6K2 is abundantly expressed in anterior horn cells of the mammalian spinal cord. We investigated the role of InsP6K2 in spinal cords of patients with amyotrophic lateral sclerosis (ALS). Autopsy specimens of lumbar spinal cords from ten patients with sporadic ALS and five non-neurological disease patients (NNDPs) were obtained. We performed quantitative real-time PCR, immunostaining, and western blotting for InsP6K1, InsP6K2, InsP6K3, protein kinase B (Akt), casein kinase 2 (CK2), and 90-kDa heat-shock protein (HSP90). In contrast to InsP6K1 and InsP6K3 mRNA expression, InsP6K2 levels in anterior horn cells of the spinal cord were significantly increased in ALS patients compared to NNDPs. In ALS patients, InsP6K2 translocated from the nucleus to the cytoplasm. However, we observed a decrease in HSP90, CK2, and Akt activity in ALS patients compared to NNDPs. A previous study reported that InsP6K2 activity is suppressed after binding to HSP90 and subsequent phosphorylation and degradation by CK2, thus decreasing InsP6K2 activity. However, InsP7, which is generated by InsP6K2, can compete with Akt for PH domain binding. Consequently, InsP7 can inhibit Akt phosphorylation. Our results suggest that InsP6K2 is activated in the spinal cord of patients with ALS and may play an important role in ALS by inducing cell death mechanisms via Akt, CK2, and HSP90 pathways.

Inositol Hexakisphosphate Kinase 2 is a Presymptomatic Biomarker for Amyotrophic Lateral Sclerosis.

OBJECTIVE: Inositol hexakisphosphate kinase 2 (InsP6K2), an enzyme that converts inositol hexakisphosphate (InsP6) to diphosphoinositol pentakisphosphate (InsP7), induces cell death. InsP6K2 is abundant in the central nervous system, especially anterior horn cells of spinal cord. To identify the role of InsP6K2 in amyotrophic lateral sclerosis (ALS), we investigated the expression levels of InsP6K2 in transgenic mice expressing mutant superoxide dismutase-1 (SOD1) (mSOD1 Tg mice). METHODS: The specimens of spinal cords were obtained from mSOD1 Tg mice and age-matched wild-type mice. We investigated the expression of InsP6K2 at the gene and protein levels of the spinal cord in mSOD1 Tg and wild-type mice. RESULTS: The gene expression levels of InsP6K2 in mSOD1 Tg mice was significantly higher than that in wild-type mice before ALS symptoms developed. In immunohistochemistry and western blotting results showed that InsP6K2 translocated from the nucleus to the cytoplasm in mSOD1 Tg mice. CONCLUSION: These findings suggest that InsP6K2 activates in mSOD1 Tg mice before the onset of ALS. Therefore, InsP6K2 might be a presymptomatic biomarker for ALS.

Inositol Hexakisphosphate Kinase 2 Promotes Cell Death in Cells with Cytoplasmic TDP-43 Aggregation.

TAR DNA-binding protein 43 (TDP-43) has been identified as a major component of ubiquitin-positive inclusions in the brains and spinal cords of patients with frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U) or amyotrophic lateral sclerosis (ALS). The phosphorylated C-terminal fragment of TDP-43 forms aggregates in the neuronal cytoplasm, possibly resulting in neuronal cell death in patients with FTLD-U or ALS. The inositol pyrophosphate known as diphosphoinositol pentakisphosphate (InsP7) contains highly energetic pyrophosphate bonds. We previously reported that inositol hexakisphosphate kinase type 2 (InsP6K2), which converts inositol hexakisphosphate (InsP6) to InsP7, mediates cell death in mammalian cells. Moreover, InsP6K2 is translocated from the nucleus to the cytosol during apoptosis. In this study, we verified that phosphorylated TDP-43 co-localized and co-bound with InsP6K2 in the cytoplasm of anterior horn cells of the spinal cord. Furthermore, we verified that cell death was augmented in the presence of cytoplasmic TDP-43 aggregations and activated InsP6K2. However, cells with only cytoplasmic TDP-43 aggregation survived because Akt activity increased. In the presence of both TDP-43 aggregation and activated InsP6K2 in the cytoplasm of cells, the expression levels of HSP90 and casein kinase 2 decreased, as the activity of Akt decreased. These conditions may promote cell death. Thus, InsP6K2 could cause neuronal cell death in patients with FTLD-U or ALS. Moreover, InsP6K2 plays an important role in a novel cell death pathway present in FTLD-U and ALS.

Possible association between dysfunction of vitamin D binding protein (GC Globulin) and migraine attacks.

To identify the genetic causality of migraine and acute, severe melalgia, we performed a linkage analysis and exome sequencing in a family with four affected individuals. We identified a variant (R21L) in exon 2 of the GC globulin gene, which is involved in the transportation of vitamin D metabolites and acts as a chemotaxic factor; this variant was co-segregated within the family. To investigate the relationship between GC globulin and melalgia, we investigated the cytokine levels in serum samples from the patients and control subjects using a cytokine antibody array. GC globulin can bind to both MCP-1 and RANTES in human serum but has a higher affinity to MCP-1. In cell culture systems, MCP-1 was able to bind to overexpressed wild-type GC globulin but not to the GC globulin variant, and the GC globulin binding affinity to MCP-1 was significantly lower in sera from the patients than in sera from control subjects. A higher concentration of MCP-1 was also observed in sera from the patients. Thus, the dysfunctional GC globulin affected cytokine release, especially the release of MCP-1, and MCP-1 might play important roles in melalgia and migraine.

Inositol hexakisphosphate kinases induce cell death in Huntington disease.

Inositol pyrophosphate diphosphoinositol pentakisphosphate is ubiquitously present in mammalian cells and contains highly energetic pyrophosphate bonds. We have previously reported that inositol hexakisphosphate kinase type 2 (InsP(6)K2), which converts inositol hexakisphosphate to inositol pyrophosphate diphosphoinositol pentakisphosphate, mediates apoptotic cell death via its translocation from the nucleus to the cytoplasm. Here, we report that InsP(6)K2 is localized mainly in the cytoplasm of lymphoblast cells from patients with Huntington disease (HD), whereas this enzyme is localized in the nucleus in control lymphoblast cells. The large number of autophagosomes detected in HD lymphoblast cells is consistent with the down-regulation of Akt in response to InsP(6)K2 activation. Consistent with these observations, the overexpression of InsP(6)Ks leads to the depletion of Akt phosphorylation and the induction of cell death. These results suggest that InsP(6)K2 activation is associated with the pathogenesis of HD.

Inositol hexakisphosphate kinases promote autophagy.

We and other authors have previously reported that increasing cellular diphosphoinositol pentakisphosphate (InsP(7)) levels increases cell sensitivity to cell death. In the present study, we elucidated the relationship between inositol hexakisphosphate kinases (InsP(6)Ks), which form InsP(7), and autophagy using InsP(6)Ks overexpression and disruption systems. A large number of autophagosomes were induced in cells transfected with InsP(6)Ks, as revealed by the conversion of LC3-I to LC3-II, which was examined using immunoblotting, immunocytochemistry, and immuno-electron microscopy for LC3; consequently, the rate of cell death was higher among these cells than among cells transfected with a control vector, as shown using propidium iodide staining. However, the reduction of InsP(6)Ks levels using RNAi suppressed the formation of autophagosomes. Moreover, the number of autophagosomes and the rate of cell death were significantly higher among cells transfected with InsP(6)Ks subjected to staurosporine-induced stress than among cells transfected with InsP(6)Ks subjected to normal conditions. The cell death induced by InsP(6)Ks was not completely suppressed by z-VAD-fmk, a pan-caspase inhibitor. The phosphorylation of mammalian target of rapamycin (mTOR) was also depressed in cells overexpressing InsP(6)Ks, suggesting that the mTOR pathway regulates autophagosomes generated by InsP(6)Ks. These findings imply that InsP(6)Ks promote autophagy and induce caspase-independent cell death. This phenomenon opens a new pathway of autophagy via InsP(6)Ks.

Role of the indigenous microbiota in maintaining the virus-specific CD8 memory T cells in the lung of mice infected with murine cytomegalovirus.

The potent role of indigenous microbiota in maintaining murine CMV (MCMV)-specific memory T cells, which were measured by multimer staining, was investigated using germfree (GF) mice. When the BALB/c mice bred under specific pathogen-free (SPF) conditions were i.p. infected with 0.2 LD(50) of MCMV, high frequencies of CD69(+)/CD44(+) MCMV-specific CD8 T cells were noted in the lungs even at 6-12 mo after infection (11.1 +/- 3.2 and 9.8 +/- 0.9%, respectively). In contrast, even though the viral load and expression levels of mRNA of such cytokines as IL-2, IL-7, IL-15, and IFN-gamma in the lungs of MCMV-infected GF mice were comparable to those of infected SPF mice, the frequencies of MCMV-specific CD8 T cells in the lungs of infected GF mice were kept lower than 1% at 6-12 mo after infection. In addition, the reconstitution of microbiota of MCMV-infected GF mice by orally administering a fecal suspension prepared from SPF mice restored the frequencies of both CD8(+)/multimer(+) and CD8(+)/multimer(-) T cells to levels similar to those found in SPF mice. These results suggested the indigenous microbiota to play a crucial role in the expansion and maintenance of viral-specific CD8 memory T cells, probably by cross-reactivity between the antigenic epitope of the MCMV-specific memory T cells and the variety of peptides derived from the members of the microbiota. Such cross-reactivity may thus be a major feature of those cells.