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.

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.

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.

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.

The neuroprotective effect of pituitary adenylate cyclase-activating polypeptide on cerebellar granule cells is mediated through inhibition of the CED3-related cysteine protease caspase-3/CPP32

Caspase-3 knockout mice exhibit thickening of the internal granule cell layer of the cerebellum. Concurrently, it has been shown that intracerebral injection of pituitary adenylate cyclase-activating polypeptide (PACAP) induces a transient increase of the thickness of the cerebellar cortex. In the present study, we have investigated the possible effect of PACAP on caspase activity in cultured cerebellar granule cells from 8-day-old rat. Incubation of granule neurons with PACAP for 24 h promoted cell survival and prevented DNA fragmentation. Exposure of cerebellar granule cells to the specific caspase-3 inhibitor N-benzyloxycarbonyl-Asp-Glu-Val-Asp fluoromethylketone (Z-DEVD-FMK) for 24 h markedly enhanced cell survival and inhibited apoptotic cell death. Time-course studies revealed that PACAP causes a prolonged inhibition of caspase-3 activity without affecting caspase-1. Administration of graded concentrations of PACAP for 3 h induced a dose-dependent inhibition of caspase-3 activity. Incubation of granule cells with both dibutyryl-cAMP (dbcAMP) and phorbol 12-myristate 13-acetate (PMA) mimicked the inhibitory effect of PACAP on caspase-3. Cotreatment of cultured neurons with the protein kinase A inhibitor H89 and the protein kinase C inhibitor chelerythrine abrogated the effect of PACAP on caspase-3 activity. In contrast, the ERK kinase inhibitor U0126 did not affect the action of PACAP on caspase-3 activity. These data demonstrate that PACAP prevents cerebellar granule neurons from apoptotic cell death through a protein kinase A- and protein kinase C-dependent inhibition of caspase-3 activity.

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.

Importance of newly generated neurons in the adult olfactory bulb for odor discrimination

In adult rodents, neurons are continually generated in the subventricular zone of the forebrain, from where they migrate tangentially toward the olfactory bulb, the only known target for these neuronal precursors. Within the main olfactory bulb, they ascend radially into the granule and periglomerular cell layers, where they differentiate mainly into local interneurons. The functional consequences of this permanent generation and integration of new neurons into existing circuits are unknown. To address this question, we used neural cell adhesion molecule-deficient mice that have documented deficits in the migration of olfactory-bulb neuron precursors, leading to about 40% size reduction of this structure. Our anatomical study reveals that this reduction is restricted to the granule cell layer, a structure that contains exclusively γ-aminobutyric acid (GABA)ergic interneurons. Furthermore, mutant mice were subjected to experiments designed to examine the behavioral consequences of such anatomical alteration. We found that the specific reduction in the newly generated interneuron population resulted in an impairment of discrimination between odors. In contrast, both the detection thresholds for odors and short-term olfactory memory were unaltered, demonstrating that a critical number of bulbar granule cells is crucial only for odor discrimination but not for general olfactory functions.

Impaired long-term potentiation induction in dentate gyrus of calretinin-deficient mice

Calretinin (Cr) is a Ca2+ binding protein present in various populations of neurons distributed in the central and peripheral nervous systems. We have generated Cr-deficient (Cr−/−) mice by gene targeting and have investigated the associated phenotype. Cr−/− mice were viable, and a large number of morphological, biochemical, and behavioral parameters were found unaffected. In the normal mouse hippocampus, Cr is expressed in a widely distributed subset of GABAergic interneurons and in hilar mossy cells of the dentate gyrus. Because both types of cells are part of local pathways innervating dentate granule cells and/or pyramidal neurons, we have explored in Cr−/− mice the synaptic transmission between the perforant pathway and granule cells and at the Schaffer commissural input to CA1 pyramidal neurons. Cr−/− mice showed no alteration in basal synaptic transmission, but long-term potentiation (LTP) was impaired in the dentate gyrus. Normal LTP could be restored in the presence of the GABAA receptor antagonist bicuculline, suggesting that in Cr−/− dentate gyrus an excess of γ-aminobutyric acid (GABA) release interferes with LTP induction. Synaptic transmission and LTP were normal in CA1 area, which contains only few Cr-positive GABAergic interneurons. Cr−/− mice performed normally in spatial memory task. These results suggest that expression of Cr contributes to the control of synaptic plasticity in mouse dentate gyrus by indirectly regulating the activity of GABAergic interneurons, and that Cr−/− mice represent a useful tool to understand the role of dentate LTP in learning and memory.

Long-term potentiation activates the GAP-43 promoter: Selective participation of hippocampal mossy cells

Perforant path long-term potentiation (LTP) in intact mouse hippocampal dentate gyrus increased the neuron-specific, growth-associated protein GAP-43 mRNA in hilar cells 3 days after tetanus, but surprisingly not in granule cells, the perforant path target. This increase was positively correlated with level of enhancement and restricted to central hilar cells on the side of stimulation. Blockade of LTP by puffing dl-aminophosphonovalerate (APV), an N-methyl-d-aspartate (NMDA) receptor blocker into the molecular layer, eliminated LTP-induced GAP-43 mRNA elevation in hilar cells. To determine whether the mRNA elevation was mediated by transcription, LTP was studied in transgenic mice bearing a GAP-43 promoter-lacZ reporter gene. Promoter activity as indexed by Transgene expression (PATE) increased as indicated by blue staining of the lacZ gene product, β-galactosidase. Potentiation induced a blue band bilaterally in the inner molecular layer of the dentate gyrus along the entire septotemporal axis. Because mossy cells are the only neurons in the central hilar zone that project to the inner molecular layer bilaterally along the entire septotemporal axis and LTP-induced activation of PATE in this zone was confined to the side of stimulation, we concluded that mossy cells were unilaterally activated, increasing synthesis of β-galactosidase, which was transported bilaterally. Neither granule cells nor pyramidal cells demonstrated increased PATE or increased GAP-43 mRNA levels. These results and recent evidence indicating the necessity of hilar neurons for LTP point to previously unheralded mossy cells as potentially critical for perforant path LTP and the GAP-43 in these cells as important for LTP persistence lasting days.

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.

Mammalian cells resistant to tumor suppressor genes.

Expression of p53 causes growth arrest or apoptosis in many normal and neoplastic cell types, but the relationship between these two effects has remained obscure. To begin to dissect the underlying mechanisms at a genetic level, we have generated mutant cells resistant to the action of wild-type p53. Rat embryo fibroblasts transformed with ras and a temperature-sensitive p53 (tsp53(135val)) gene were chemically mutagenized and selected for growth at a temperature at which p53 adopts a wild-type conformation (31.5 degrees C). Clones that grew exponentially at 31.5 degrees C were selected. Cell fusion experiments demonstrated that the mutations conferring resistance to p53-mediated growth arrest were dominant. The mutagenized clones were resistant not only to p53-mediated growth arrest, but also to the apoptosis induced by E1A in conjunction with p53, and partially resistant to the retinoblastoma tumor suppressor, pRB. The results suggest that a single downstream pathway can control the induction of growth arrest and apoptosis, and that both p53 and RB function through this pathway.

The ALIAmide palmitoylethanolamide and cannabinoids, but not anandamide, are protective in a delayed postglutamate paradigm of excitotoxic death in cerebellar granule neurons.

The amino acid L-glutamate is a neurotransmitter that mediates fast neuronal excitation in a majority of synapses in the central nervous system. Glutamate stimulates both N-methyl-D-aspartate (NMDA) and non-NMDA receptors. While activation of NMDA receptors has been implicated in a variety of neurophysiologic processes, excessive NMDA receptor stimulation (excitotoxicity) is thought to be primarily responsible for neuronal injury in a wide variety of acute neurological disorders including hypoxia-ischemia, seizures, and trauma. Very little is known about endogenous molecules and mechanisms capable of modulating excitotoxic neuronal death. Saturated N-acylethanolamides like palmitoylethanolamide accumulate in ischemic tissues and are synthesized by neurons upon excitatory amino acid receptor activation. Here we report that palmitoylethanolamide, but not the cognate N-acylamide anandamide (the ethanolamide of arachidonic acid), protects cultured mouse cerebellar granule cells against glutamate toxicity in a delayed postagonist paradigm. Palmitoylethanolamide reduced this injury in a concentration-dependent manner and was maximally effective when added 15-min postglutamate. Cannabinoids, which like palmitoylethanolamide are functionally active at the peripheral cannabinoid receptor CB2 on mast cells, also prevented neuron loss in this delayed postglutamate model. Furthermore, the neuroprotective effects of palmitoylethanolamide, as well as that of the active cannabinoids, were efficiently antagonized by the candidate central cannabinoid receptor (CB1) agonist anandamide. Analogous pharmacological behaviors have been observed for palmitoylethanolamide (ALI-Amides) in downmodulating mast cell activation. Cerebellar granule cells expressed mRNA for CB1 and CB2 by in situ hybridization, while two cannabinoid binding sites were detected in cerebellar membranes. The results suggest that (i) non-CB1 cannabinoid receptors control, upon agonist binding, the downstream consequences of an excitotoxic stimulus; (ii) palmitoylethanolamide, unlike anandamide, behaves as an endogenous agonist for CB2-like receptors on granule cells; and (iii) activation of such receptors may serve to downmodulate deleterious cellular processes following pathological events or noxious stimuli in both the nervous and immune systems.

Synaptic activation of NF-kappa B by glutamate in cerebellar granule neurons in vitro.

Neuronal proliferation, migration, and differentiation are regulated by the sequential expression of particular genes at specific stages of development. Such processes rely on differential gene expression modulated through second-messenger systems. Early postnatal mouse cerebellar granule cells migrate into the internal granular layer and acquire differentiated properties. The neurotransmitter glutamate has been shown to play an important role in this developmental process. We show here by immunohistochemistry that the RelA subunit of the transcription factor NF-kappa B is present in several areas of the mouse brain. Moreover, immunofluorescence microscopy and electrophoretic mobility-shift assay demonstrate that in cerebellar granule cell cultures derived from 3- to 7-day-old mice, glutamate specifically activates the transcription factor NF-kappa B, as shown by binding of nuclear extract proteins to a synthetic oligonucleotide reproducing the kappa B site of human immunodeficiency virus. The use of different antagonists of the glutamate recpetors indicates that the effect of glutamate occurs mainly via N-methyl-D-aspartate (NMDA)-receptor activation, possibly as a result of an increase in intracellular Ca2+. The synaptic specificity of the effect is strongly suggested by the observation that glutamate failed to activate NF-kappa B in astrocytes, while cytokines, such as interleukin 1 alpha and tumor necrosis factor alpha, did so. The effect of glutamate appears to be developmentally regulated. Indeed, NF-kappa B is found in an inducible form in the cytoplasm of neurons of 3- to 7-day-old mice but is constitutively activated in the nuclei of neurons derived from older pups (8-10 days postnatal). Overall, these observations suggest the existence of a new pathway of trans-synaptic regulation of gene expression.

Long-term potentiation and dual-component quantal signaling in the dentate gyrus

Long-term potentiation (LTP) of excitatory transmission is an important candidate cellular mechanism for the storage of memories in the mammalian brain. The subcellular phenomena that underlie the persistent increase in synaptic strength, however, are incompletely understood. A potentially powerful method to detect a presynaptic increase in glutamate release is to examine the effect of LTP induction on the rate at which the use-dependent blocker MK-801 attenuates successive N-methyl-d-aspartic acid (NMDA) receptor-mediated synaptic signals. This method, however, has given apparently contradictory results when applied in hippocampal CA1. The inconsistency could be explained if NMDA receptors were opened by glutamate not only released from local presynaptic terminals, but also diffusing from synapses on neighboring cells where LTP was not induced. Here we examine the effect of pairing-induced LTP on the MK-801 blocking rate in two afferent inputs to dentate granule cells. LTP in the medial perforant path is associated with a significant increase in the MK-801 blocking rate, implying a presynaptic increase in glutamate release probability. An enhanced MK-801 blocking rate is not seen, however, in the lateral perforant path. This result still could be compatible with a presynaptic contribution to LTP in the lateral perforant path if intersynaptic cross-talk occurred. In support of this hypothesis, we show that NMDA receptors consistently sense more quanta of glutamate than do α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors. In the medial perforant path, in contrast, there is no significant difference in the number of quanta mediated by the two receptors. These results support a presynaptic contribution to LTP and imply that differences in intersynaptic cross-talk can complicate the interpretation of experiments designed to detect changes in transmitter release.

Activity-dependent regulation of synaptic clustering in a hippocampal culture system

Currently, there is a limited understanding of the factors that influence the localization and density of individual synapses in the central nervous system. Here we have studied the effects of activity on synapse formation between hippocampal dentate granule cells and CA3 pyramidal neurons in culture, taking advantage of FM1–43 as a fluorescent marker of synaptic boutons. We observed an early tendency for synapses to group together, quickly followed by the appearance of synaptic clusters on dendritic processes. These events were strongly influenced by N-methyl-d-aspartic acid receptor- and cyclic AMP-dependent signaling. The microstructure and localization of the synaptic clusters resembled that found in hippocampus, at mossy fiber synapses of stratum lucidum. Activity-dependent clustering of synapses represents a means for synaptic targeting that might contribute to synaptic organization in the brain.