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.

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.

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.

Characterization of mutant mice that express polyglutamine in cerebellar Purkinje cells.

We recently produced transgenic mice that expressed an abnormally expanded polyglutamine (polyQ) specifically in cerebellar Purkinje cells (polyQ mice). The polyQ mice showed inclusion body formation, cerebellar atrophy and severe ataxia. Here we analyzed polyQ mice using immunohistochemistry, immunoelectronmicroscopy and electrophysiology. A diffuse form of polyQ was detected in the nucleus. Interestingly, ubiquitinated large inclusions were located close to, but apparently outside of the soma of Purkinje cells. Infusion of lucifer yellow into Purkinje cells clearly indicated the traffic between the periplasmic inclusions and soma of Purkinje cells. To examine whether the formation of periplasmic inclusions was an active process or a result of cell death, the polyQ mouse cerebellum was immunolabeled for cleaved caspase-3, a marker of apoptosis. Interestingly, no Purkinje cells in P80 polyQ mice immunoreacted with the antibody. The results were substantiated by electrophysiological assay, which showed that P80 Purkinje cells with large periplasmic inclusions were functionally active: excitatory postsynaptic currents (EPSCs) were reliably evoked upon electrical stimulation of parallel fibers (PFs) or climbing fibers (CFs), and current injection into Purkinje cells generated action potentials; however, the frequency of action potentials in response to various volumes of current injection was consistently lower in polyQ mice than in wild-type animals, and aberrant innervation by multiple CFs was detected in polyQ mouse Purkinje cells. These results suggest that Purkinje cells with periplasmic inclusions were not apoptotic, but their functions were substantially impaired, which could contribute to the severe ataxic phenotype.

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.