The PtdIns(3,4)P(2) phosphatase INPP4A is a suppressor of excitotoxic neuronal death.

Phosphorylated derivatives of phosphatidylinositol, collectively referred to as phosphoinositides, occur in the cytoplasmic leaflet of cellular membranes and regulate activities such as vesicle transport, cytoskeletal reorganization and signal transduction. Recent studies have indicated an important role for phosphoinositide metabolism in the aetiology of diseases such as cancer, diabetes, myopathy and inflammation. Although the biological functions of the phosphatases that regulate phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) have been well characterized, little is known about the functions of the phosphatases regulating the closely related molecule phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P(2)). Here we show that inositol polyphosphate phosphatase 4A (INPP4A), a PtdIns(3,4)P(2) phosphatase, is a suppressor of glutamate excitotoxicity in the central nervous system. Targeted disruption of the Inpp4a gene in mice leads to neurodegeneration in the striatum, the input nucleus of the basal ganglia that has a central role in motor and cognitive behaviours. Notably, Inpp4a(-/-) mice show severe involuntary movement disorders. In vitro, Inpp4a gene silencing via short hairpin RNA renders cultured primary striatal neurons vulnerable to cell death mediated by N-methyl-d-aspartate-type glutamate receptors (NMDARs). Mechanistically, INPP4A is found at the postsynaptic density and regulates synaptic NMDAR localization and NMDAR-mediated excitatory postsynaptic current. Thus, INPP4A protects neurons from excitotoxic cell death and thereby maintains the functional integrity of the brain. Our study demonstrates that PtdIns(3,4)P(2), PtdIns(3,4,5)P(3) and the phosphatases acting on them can have distinct regulatory roles, and provides insight into the unique aspects and physiological significance of PtdIns(3,4)P(2) metabolism. INPP4A represents, to our knowledge, the first signalling protein with a function in neurons to suppress excitotoxic cell death. The discovery of a direct link between PtdIns(3,4)P(2) metabolism and the regulation of neurodegeneration and involuntary movements may aid the development of new approaches for the treatment of neurodegenerative disorders.

High transgene expression by lentiviral vectors causes maldevelopment of Purkinje cells in vivo.

Lentiviral vectors are promising as gene-transfer vehicles for gene therapy targeted to intractable brain diseases. Although lentiviral vectors are thought to exert little toxicity on infected cells, the adverse influence of viral infection on vulnerable developing neurons has not been well studied. Here, we examined whether lentiviral vector infection and subsequent transgene expression affected the morphological and functional maturation of vigorously developing cerebellar Purkinje cells in vivo. Lentiviral vectors expressing GFP under the control of the murine stem cell virus (MSCV) promoter were injected into the cerebellar cortex of neonatal rat pups. Three weeks after treatment, GFP-expressing Purkinje cells were compared with control Purkinje cells from phosphate-buffered saline-injected rats. Analysis of the dendritic tree showed that total dendrite length in GFP-expressing Purkinje cells was almost 80% that in control Purkinje cells. Electrophysiological examination showed that short-term synaptic plasticity at parallel fiber-Purkinje cell synapses and climbing fiber-Purkinje cell synapses was significantly altered in GFP-expressing Purkinje cells. In contrast, maldevelopment of infected Purkinje cells was substantially attenuated when lentiviral vectors with much weaker promoter activity were used. These results suggest that the maldevelopment of Purkinje cells was mainly caused by subsequent expression of a high amount of GFP driven by the strong MSCV promoter. Thus, the use of lentiviral vectors carrying a strong promoter may require particular precautions when applying them to neurological disorders of infants.

Transgene expression in the mouse cerebellar Purkinje cells with a minimal level of integration using long terminal repeat-modified lentiviral vectors.

Lentiviral vectors (LVs), which preferentially target nondividing cells, such as neurons, are promising tools for gene therapy. However, these vectors are still unsuitable as they result in insertional mutagenesis. It is therefore essential to prevent insertional mutagenesis if these vectors are to be adopted for safe next generation clinical applications. In order to establish safe genetic therapy with LVs, we focused on the integrase recognition sequence (att) in the long terminal repeat (LTR), which is localized at the edge of the preintegrated viral DNA. We generated LTR-modified LVs (LMLVs), by altering the conserved sequences located just before the cleavage site; this alteration prevented the integration of viral DNA into the host genome. In this study, the LMLVs significantly decreased the LV-mediated transgene expression in HeLa cells compared to the control, i.e., wild-type LTR LVs; this supposedly occurred because integration was prevented. In addition, LMLVs exhibited gene expression in vivo when they were injected into the mouse cerebellum. Moreover, quantitative Alu element-mediated polymerase chain reaction (Alu-PCR), which detects integrated viral DNA, revealed that rate of LMLV-suppressed integration was approximately 1/500-fold compared to that in the case of the wild-type LTR LV. These data suggest that LMLVs efficiently prevent integration as well as exhibit LV-mediated gene expression in mouse cerebellar Purkinje cells in vivo.

Lentiviral vector-mediated rescue of motor behavior in spontaneously occurring hereditary ataxic mice.

Hotfoot5J mice are spontaneously occurring ataxic mice that lack delta2 glutamate receptor (GluRdelta2) protein in cerebellar Purkinje cells. Here we aimed to rescue the ataxic phenotype of hotfoot5J mice by lentiviral vector-mediated expression of recombinant GluRdelta2 in Purkinje cells. Lentiviral vectors expressing GluRdelta2 were injected into the cerebellar cortex of hotfoot5J mice 6 or 7 days after birth, and the effects were studied on postnatal day 30. The motor behavior of hotfoot5J mice treated with vectors expressing GluRdelta2 was markedly rescued, whereas the ataxia of hotfoot5J mice treated with vectors expressing GFP was comparable to that of non-injected hotfoot5J littermates. Furthermore, the impaired release probability of glutamate from parallel fiber terminals and the failure of developmental elimination of surplus climbing fibers from Purkinje cells in hotfoot5J mice were completely rescued by GluRdelta2 expression. These results indicate the therapeutic potential of viral vector-based gene therapy for hereditary cerebellar ataxia and other neuronal disorders.

Rescue of abnormal phenotypes in delta2 glutamate receptor-deficient mice by the extracellular N-terminal and intracellular C-terminal domains of the delta2 glutamate receptor.

The delta2 glutamate receptor (GluRdelta2) is expressed predominantly in cerebellar Purkinje cells. GluRdelta2 knock-out mice show impaired synaptogenesis and loss of long-term depression (LTD) at parallel fiber/Purkinje cell synapses, and persistent multiple climbing fiber (CF) innervation of Purkinje cells, resulting in severe ataxia. To identify domains critical for GluRdelta2 function, we produced various GluRdelta2 deletion constructs. Using lentiviral vectors, those constructs were expressed in Purkinje cells of GluRdelta2-deficient mice at postnatal day (P) 6 or 7, and rescue of abnormal phenotypes was examined beyond P30. Most constructs failed to rescue the defects of GluRdelta2-deficient mice, mainly because they were not efficiently transferred to the postsynaptic sites. However, a construct carrying only the extracellular N-terminal domain (NTD) and the intracellular C-terminal domain (CTD) linked with the fourth transmembrane domain of GluRdelta2 (NTD-TM4-CTD) caused incomplete, but significant rescue of ataxia, consistent with relatively better transport of the construct to the synapses. Notably, the expression of NTD-TM4-CTD in GluRdelta2-deficient Purkinje cells restored abrogated LTD, and aberrant CF territory in the molecular layer. Although the expression of NTD-TM4-CTD failed to rescue persistent multiple CF innervation of GluRdelta2-deficient Purkinje cells, a similar construct in which only TM4 was replaced with a transmembrane domain of CD4 successfully rescued the multiple CF innervation, probably due to more efficient transport of the protein to postsynaptic sites. These results suggest that NTD and CTD are critical domains of GluRdelta2, which functions substantially without conventional ligand binding and ion channel structures.

Purkinje-cell-preferential transduction by lentiviral vectors with the murine stem cell virus promoter.

Viral-vector-mediated gene delivery into Purkinje cells is a promising method for exploring the pathophysiology of the cerebellum; however, it is generally difficult to achieve sufficiently high levels of gene expression in Purkinje cells using viral vectors with a cell-type-specific promoter because of the weakness of transcriptional activity. In this study, we prepared lentiviral vectors that express GFP under the control of various ubiquitous promoters derived from murine stem cell virus (MSCV), cytomegalovirus (CMV), CMV early enhancer/chicken beta actin (CAG), and Rous sarcoma virus (RSV) and compared their potential to transduce Purkinje cells. Mice were sacrificed 7 days after lentiviral injection and the ratios of GFP(+) Purkinje cells to all transduced cells were determined. The highest transduction ratio was observed when we used lentivectors containing the MSCV promoter: approximately 70% of GFP(+) cells were Purkinje cells, the next highest ratio was for the CMV promoter (approximately 40%), then the CAG promoter (approximately 35%), and the lowest ratio was for the RSV promoter (approximately 10%). Moreover, the highest levels of GFP expression were also caused by the MSCV promoter. Thus, among the ubiquitous promoters examined, the MSCV promoter was the best for the expression of a foreign gene in Purkinje cells in vivo.

Lentivector-mediated rescue from cerebellar ataxia in a mouse model of spinocerebellar ataxia.

Polyglutamine disorders are inherited neurodegenerative diseases caused by the accumulation of expanded polyglutamine protein (polyQ). Previously, we identified a new guanosine triphosphatase, CRAG, which facilitates the degradation of polyQ aggregates through the ubiquitin-proteasome pathway in cultured cells. Because expression of CRAG decreases in the adult brain, a reduced level of CRAG could underlie the onset of polyglutamine diseases. To examine the potential of CRAG expression for treating polyglutamine diseases, we generated model mice expressing polyQ predominantly in Purkinje cells. The model mice showed poor dendritic arborization of Purkinje cells, a markedly atrophied cerebellum and severe ataxia. Lentivector-mediated expression of CRAG in Purkinje cells of model mice extensively cleared polyQ aggregates and re-activated dendritic differentiation, resulting in a striking rescue from ataxia. Our in vivo data substantiate previous cell-culture-based results and extend further the usefulness of targeted delivery of CRAG as a gene therapy for polyglutamine diseases.