Loss of p21 increases sensitivity to ionizing radiation and delays the onset of lymphoma in atm-deficient mice

Ataxia telangiectasia (AT) is an autosomal recessive disorder characterized by growth retardation, cerebellar ataxia, oculocutaneous telangiectasias, and a high incidence of lymphomas and leukemias. In addition, AT patients are sensitive to ionizing radiation. Atm-deficient mice recapitulate most of the AT phenotype. p21cip1/waf1 (p21 hereafter), an inhibitor of cyclin-dependent kinases, has been implicated in cellular senescence and response to γ-radiation-induced DNA damage. To study the role of p21 in ATM-mediated signal transduction pathways, we examined the combined effect of the genetic loss of atm and p21 on growth control, radiation sensitivity, and tumorigenesis. As might have been expected, our data provide evidence that p21 modifies the in vitro senescent response seen in AT fibroblasts. Further, it is a downstream effector of ATM-mediated growth control. In addition, however, we find that loss of p21 in the context of an atm-deficient mouse leads to a delay in thymic lymphomagenesis and an increase in acute radiation sensitivity in vivo (the latter principally because of effects on the gut epithelium). Modification of these two crucial aspects of the ATM phenotype can be related to an apparent increase in spontaneous apoptosis seen in tumor cells and in the irradiated intestinal epithelium of mice doubly null for atm and p21. Thus, loss of p21 seems to contribute to tumor suppression by a mechanism that operates via a sensitized apoptotic response. These results have implications for cancer therapy in general and AT patients in particular.

Ablation of P/Q-type Ca2+ channel currents, altered synaptic transmission, and progressive ataxia in mice lacking the α1A-subunit

The Ca2+ channel α1A-subunit is a voltage-gated, pore-forming membrane protein positioned at the intersection of two important lines of research: one exploring the diversity of Ca2+ channels and their physiological roles, and the other pursuing mechanisms of ataxia, dystonia, epilepsy, and migraine. α1A-Subunits are thought to support both P- and Q-type Ca2+ channel currents, but the most direct test, a null mutant, has not been described, nor is it known which changes in neurotransmission might arise from elimination of the predominant Ca2+ delivery system at excitatory nerve terminals. We generated α1A-deficient mice (α1A−/−) and found that they developed a rapidly progressive neurological deficit with specific characteristics of ataxia and dystonia before dying ≈3–4 weeks after birth. P-type currents in Purkinje neurons and P- and Q-type currents in cerebellar granule cells were eliminated completely whereas other Ca2+ channel types, including those involved in triggering transmitter release, also underwent concomitant changes in density. Synaptic transmission in α1A−/− hippocampal slices persisted despite the lack of P/Q-type channels but showed enhanced reliance on N-type and R-type Ca2+ entry. The α1A−/− mice provide a starting point for unraveling neuropathological mechanisms of human diseases generated by mutations in α1A.

Accumulation of protease-resistant prion protein (PrP) and apoptosis of cerebellar granule cells in transgenic mice expressing a PrP insertional mutation

We have generated lines of transgenic mice that express a mutant prion protein (PrP) containing 14 octapeptide repeats whose human homologue is associated with an inherited prion dementia. These mice develop a neurological illness with prominent ataxia at 65 or 240 days of age, depending on whether the transgene array is, respectively, homozygous or hemizygous. Starting from birth, mutant PrP is converted into a protease-resistant and detergent-insoluble form that resembles the scrapie isoform of PrP, and this form accumulates dramatically in many brain regions throughout the lifetime of the mice. As PrP accumulates, there is massive apoptosis of granule cells in the cerebellum. Our analysis provides important insights into the molecular pathogenesis of inherited prion disorders in humans.

Impaired motor coordination and persistent multiple climbing fiber innervation of cerebellar Purkinje cells in mice lacking Gαq

Mice lacking the α-subunit of the heterotrimeric guanine nucleotide binding protein Gq (Gαq) are viable but suffer from ataxia with typical signs of motor discoordination. The anatomy of the cerebellum is not overtly disturbed, and excitatory synaptic transmission from parallel fibers to cerebellar Purkinje cells (PCs) and from climbing fibers (CFs) to PCs is functional. However, about 40% of adult Gαq mutant PCs remain multiply innervated by CFs because of a defect in regression of supernumerary CFs in the third postnatal week. Evidence is provided suggesting that Gαq is part of a signaling pathway that is involved in the elimination of multiple CF innervation during this period.

The weaver mutation of GIRK2 results in a loss of inwardly rectifying K+ current in cerebellar granule cells.

The weaver mutation in mice results in a severe ataxia that is attributable to the degeneration of cerebellar granule cells and dopaminergic neurons in the substantia nigra. Recent genetic studies indicate that the GIRK2 gene is altered in weaver. This gene codes for a G-protein-activated, inwardly rectifying K+ channel protein (8). The mutation results in a single amino acid substitution (glycine-->serine) in the pore-forming H5 region of the channel. The functional consequences of this mutation appear to depend upon the co-expression of other GIRK subunits--leading to either a gain or loss of function. Here, we show that G-protein-activated inwardly rectifying K+ currents are significantly reduced in cerebellar granule cells from animals carrying the mutant allele. The reduction is most pronounced in homozygous neurons. These findings suggest that the death of neurons in weaver is attributable to the loss of GIRK2-mediated currents, not to the expression of a nonspecific cation current.

KARP-1 is induced by DNA damage in a p53- and ataxia telangiectasia mutated-dependent fashion

The KARP-1 (Ku86 Autoantigen Related Protein-1) gene, which is expressed from the human Ku86 autoantigen locus, appears to play a role in mammalian DNA double-strand break repair as a regulator of the DNA-dependent protein kinase complex. Here we demonstrate that KARP-1 gene expression is significantly up-regulated following exposure of cells to DNA damage. KARP-1 mRNA induction was completely dependent on the ataxia telangiectasia and p53 gene products, consistent with the presence of a p53 binding site within the second intron of the KARP-1 locus. These observations link ataxia telangiectasia, p53, and KARP-1 in a common pathway.

Antisense oligonucleotides against α1E reduce R-type calcium currents in cerebellar granule cells

Many neurons of the central nervous system display multiple high voltage-activated Ca2+ currents, pharmacologically classified as L-, N-, P-, Q-, and R-type. Of these current types, the R-type is the least understood. The leading candidate for the molecular correlate of R-type currents in cerebellar granule cells is the α1E subunit, which yields Ca2+ currents very similar to the R-type when expressed in heterologous systems. As a complementary approach, we tested whether antisense oligonucleotides against α1E could decrease the expression of R-type current in rat cerebellar granule neurons in culture. Cells were supplemented with either antisense or sense oligonucleotides and whole-cell patch clamp recordings were obtained after 6–8 days in vitro. Incubation with α1E antisense oligonucleotide caused a 52.5% decrease in the peak R-type current density, from −10 ± 0.6 picoamperes/picofarad (pA/pF) (n = 6) in the untreated controls to −4.8 ± 0.8 pA/pF (n = 11) (P < 0.01). In contrast, no significant changes in the current expression were seen in sense oligonucleotide-treated cells (−11.3 ± 3.2 pA/pF). The specificity of the α1E antisense oligonucleotides was supported by the lack of change in estimates of the P/Q current amplitude. Furthermore, antisense and sense oligonucleotides against α1A did not affect R-type current expression (−11.5 ± 1.7 and −11.7 ± 1.7 pA/pF, respectively), whereas the α1A antisense oligonucleotide significantly reduced whole cell currents under conditions in which P/Q current is dominant. Our results support the hypothesis that members of the E class of α1 subunits support the high voltage-activated R-type current in cerebellar granule cells.

Selective loss of dopaminergic nigro-striatal neurons in brains of Atm-deficient mice

Ataxia-telangiectasia (AT) is a human disease caused by mutations in the ATM gene. The neural phenotype of AT includes progressive cerebellar neurodegeneration, which results in ataxia and eventual motor dysfunction. Surprisingly, mice in which the Atm gene has been inactivated lack distinct behavioral ataxia or pronounced cerebellar degeneration, the hallmarks of the human disease. To determine whether lack of the Atm protein can nonetheless lead to structural abnormalities in the brain, we compared brains from male Atm-deficient mice with male, age-matched controls. Atm-deficient mice exhibited severe degeneration of tyrosine hydroxylase-positive, dopaminergic nigro-striatal neurons, and their terminals in the striatum. This cell loss was accompanied by a large reduction in immunoreactivity for the dopamine transporter in the striatum. A reduction in dopaminergic neurons also was evident in the ventral tegmental area. This effect was selective in that the noradrenergic nucleus locus coeruleus was normal in these mice. Behaviorally, Atm-deficient mice expressed locomotor abnormalities manifested as stride-length asymmetry, which could be corrected by peripheral application of the dopaminergic precursor l-dopa. In addition, these mice were hypersensitive to the dopamine releasing drug d-amphetamine. These results indicate that ATM deficiency can severely affect dopaminergic neurons in the central nervous system and suggest possible strategies for treating this aspect of the disease.

Directing gene expression to cerebellar granule cells using γ-aminobutyric acid type A receptor α6 subunit transgenes

Expression of the γ-aminobutyric acid type A receptor α6 subunit gene is restricted to differentiated granule cells of the cerebellum and cochlear nucleus. The mechanisms underlying this limited expression are unknown. Here we have characterized the expression of a series of α6-based transgenes in adult mouse brain. A DNA fragment containing a 1-kb portion upstream of the start site(s), together with exons 1–8, can direct high-level cerebellar granule cell-specific reporter gene expression. Thus powerful granule cell-specific determinants reside within the 5′ half of the α6 subunit gene body. This intron-containing transgene appears to lack the cochlear nucleus regulatory elements. It therefore provides a cassette to deliver gene products solely to adult cerebellar granule cells.

Activation of a caspase 3-related cysteine protease is required for glutamate-mediated apoptosis of cultured cerebellar granule neurons

Neurotoxicity induced by overstimulation of N-methyl-d-aspartate (NMDA) receptors is due, in part, to a sustained rise in intracellular Ca2+; however, little is known about the ensuing intracellular events that ultimately result in cell death. Here we show that overstimulation of NMDA receptors by relatively low concentrations of glutamate induces apoptosis of cultured cerebellar granule neurons (CGNs) and that CGNs do not require new RNA or protein synthesis. Glutamate-induced apoptosis of CGNs is, however, associated with a concentration- and time-dependent activation of the interleukin 1β-converting enzyme (ICE)/CED-3-related protease, CPP32/Yama/apopain (now designated caspase 3). Further, the time course of caspase 3 activation after glutamate exposure of CGNs parallels the development of apoptosis. Moreover, glutamate-induced apoptosis of CGNs is almost completely blocked by the selective cell permeable tetrapeptide inhibitor of caspase 3, Ac-DEVD-CHO but not by the ICE (caspase 1) inhibitor, Ac-YVAD-CHO. Western blots of cytosolic extracts from glutamate-exposed CGNs reveal both cleavage of the caspase 3 substrate, poly(ADP-ribose) polymerase, as well as proteolytic processing of pro-caspase 3 to active subunits. Our data demonstrate that glutamate-induced apoptosis of CGNs is mediated by a posttranslational activation of the ICE/CED-3-related cysteine protease caspase 3.

Defective γ-aminobutyric acid type B receptor-activated inwardly rectifying K+ currents in cerebellar granule cells isolated from weaver and Girk2 null mutant mice

Stimulation of inhibitory neurotransmitter receptors, such as γ-aminobutyric acid type B (GABAB) receptors, activates G protein-gated inwardly rectifying K+ channels (GIRK) which, in turn, influence membrane excitability. Seizure activity has been reported in a Girk2 null mutant mouse lacking GIRK2 channels but showing normal cerebellar development as well as in the weaver mouse, which has mutated GIRK2 channels and shows abnormal development. To understand how the function of GIRK2 channels differs in these two mutant mice, we compared the G protein-activated inwardly rectifying K+ currents in cerebellar granule cells isolated from Girk2 null mutant and weaver mutant mice with those from wild-type mice. Activation of GABAB receptors in wild-type granule cells induced an inwardly rectifying K+ current, which was sensitive to pertussis toxin and inhibited by external Ba2+ ions. The amplitude of the GABAB receptor-activated current was severely attenuated in granule cells isolated from both weaver and Girk2 null mutant mice. By contrast, the G protein-gated inwardly rectifying current and possibly the agonist-independent basal current appeared to be less selective for K+ ions in weaver but not Girk2 null mutant granule cells. Our results support the hypothesis that a nonselective current leads to the weaver phenotype. The loss of GABAB receptor-activated GIRK current appears coincident with the absence of GIRK2 channel protein and the reduction of GIRK1 channel protein in the Girk2 null mutant mouse, suggesting that GABAB receptors couple to heteromultimers composed of GIRK1 and GIRK2 channel subunits.

Evolution of the Friedreich’s ataxia trinucleotide repeat expansion: Founder effect and premutations

Friedreich’s ataxia, the most frequent inherited ataxia, is caused, in the vast majority of cases, by large GAA repeat expansions in the first intron of the frataxin gene. The normal sequence corresponds to a moderately polymorphic trinucleotide repeat with bimodal size distribution. Small normal alleles have approximately eight to nine repeats whereas a more heterogeneous mode of large normal alleles ranges from 16 to 34 GAA. The latter class accounts for ≈17% of normal alleles. To identify the origin of the expansion mutation, we analyzed linkage disequilibrium between expansion mutations or normal alleles and a haplotype of five polymorphic markers within or close to the frataxin gene; 51% of the expansions were associated with a single haplotype, and the other expansions were associated with haplotypes that could be related to the major one by mutation at a polymorphic marker or by ancient recombination. Of interest, the major haplotype associated with expansion is also the major haplotype associated with the larger alleles in the normal size range and was almost never found associated with the smaller normal alleles. The results indicate that most if not all large normal alleles derive from a single founder chromosome and that they represent a reservoir for larger expansion events, possibly through “premutation” intermediates. Indeed, we found two such alleles (42 and 60 GAA) that underwent cataclysmic expansion to pathological range in a single generation. This stepwise evolution to large trinucleotide expansions already was suggested for myotonic dystrophy and fragile X syndrome and may relate to a common mutational mechanism, despite sequence motif differences.

Pleiotropic defects in ataxia-telangiectasia protein-deficient mice

We have generated a mouse model for ataxia-telangiectasia by using gene targeting to generate mice that do not express the Atm protein. Atm-deficient mice are retarded in growth, do not produce mature sperm, and exhibit severe defects in T cell maturation while going on to develop thymomas. Atm-deficient fibroblasts grow poorly in culture and display a high level of double-stranded chromosome breaks. Atm-deficient thymocytes undergo spontaneous apoptosis in vitro significantly more than controls. Atm-deficient mice then exhibit many of the same symptoms found in ataxia-telangiectasia patients and in cells derived from them. Furthermore, we demonstrate that the Atm protein exists as two discrete molecular species, and that loss of one or of both of these can lead to the development of the disease.

Neuronal migration is retarded in mice lacking the tissue plasminogen activator gene

Neuronal migration is a critical phase of brain development, where defects can lead to severe ataxia, mental retardation, and seizures. In the developing cerebellum, granule neurons turn on the gene for tissue plasminogen activator (tPA) as they begin their migration into the cerebellar molecular layer. Granule neurons both secrete tPA, an extracellular serine protease that converts the proenzyme plasminogen into the active protease plasmin, and bind tPA to their cell surface. In the nervous system, tPA activity is correlated with neurite outgrowth, neuronal migration, learning, and excitotoxic death. Here we show that compared with their normal counterparts, mice lacking the tPA gene (tPA−/−) have greater than 2-fold more migrating granule neurons in the cerebellar molecular layer during the most active phase of granule cell migration. A real-time analysis of granule cell migration in cerebellar slices of tPA−/− mice shows that granule neurons are migrating 51% as fast as granule neurons in slices from wild-type mice. These findings establish a direct role for tPA in facilitating neuronal migration, and they raise the possibility that late arriving neurons may have altered synaptic interactions.

Patches of synchronized activity in the cerebellar cortex evoked by mossy-fiber stimulation: Questioning the role of parallel fibers

The discrepancy between the structural longitudinal organization of the parallel-fiber system in the cerebellar cortex and the functional mosaic-like organization of the cortex has provoked controversial theories about the flow of information in the cerebellum. We address this issue by characterizing the spatiotemporal organization of neuronal activity in the cerebellar cortex by using optical imaging of voltage-sensitive dyes in isolated guinea-pig cerebellum. Parallel-fiber stimulation evoked a narrow beam of activity, which propagated along the parallel fibers. Stimulation of the mossy fibers elicited a circular, nonpropagating patch of synchronized activity. These results strongly support the hypothesis that a beam of parallel fibers, activated by a focal group of granule cells, fails to activate the Purkinje cells along most of its length. It is thus the ascending axon of the granule cell, and not its parallel branches, that activates and defines the basic functional modules of the cerebellar cortex.