Organotypic Cerebellar Slice Research Articles

The Plasma Membrane Ca2+-ATPase2 (PMCA2) Is Involved in the Regulation of Purkinje Cell Dendritic Growth in Cerebellar Organotypic Slice Cultures

Purkinje cells are the principal neurons of the cerebellar cortex and have an extensive and elaborate dendritic tree. Chronic activation of type I metabotropic glutamate receptors inhibits Purkinje cell dendritic growth in organotypic cerebellar slice cultures. This effect is mediated by calcium influx through P/Q-type and T-type Ca2+ channels. We have now studied the role of the plasma membrane Ca2+-ATPase2 (PMCA2), a major calcium extrusion pump, for Purkinje cell dendritic development. We found that PMCA2 is strongly expressed in the plasma membrane and dendritic spines of Purkinje cells in organotypic slice cultures compatible with a role for controlling the local dendritic calcium equilibrium. Inhibition of PMCA2 activity by carboxyeosin resulted in a moderate reduction of Purkinje cell dendritic tree size indicating that the extrusion of calcium by PMCA2 is important for maintaining the dendritic calcium concentration and controlling dendritic growth. When inhibition of PMCA2 was combined with stimulation of type I metabotropic glutamate receptors, it partially rescued dendritic morphology. This protection can be explained by a compensatory inactivation of voltage-gated calcium channels in Purkinje cells after PMCA2 inhibition. Our results demonstrate that PMCA2 activity is an important regulator of the dendritic calcium equilibrium controlling Purkinje cell dendritic growth.

Prion pathogenesis is faithfully reproduced in cerebellar organotypic slice cultures.

Prions cause neurodegeneration in vivo, yet prion-infected cultured cells do not show cytotoxicity. This has hampered mechanistic studies of prion-induced neurodegeneration. Here we report that prion-infected cultured organotypic cerebellar slices (COCS) experienced progressive spongiform neurodegeneration closely reproducing prion disease, with three different prion strains giving rise to three distinct patterns of prion protein deposition. Neurodegeneration did not occur when PrP was genetically removed from neurons, and a comprehensive pharmacological screen indicated that neurodegeneration was abrogated by compounds known to antagonize prion replication. Prion infection of COCS and mice led to enhanced fodrin cleavage, suggesting the involvement of calpains or caspases in pathogenesis. Accordingly, neurotoxicity and fodrin cleavage were prevented by calpain inhibitors but not by caspase inhibitors, whereas prion replication proceeded unimpeded. Hence calpain inhibition can uncouple prion replication from its neurotoxic sequelae. These data validate COCS as a powerful model system that faithfully reproduces most morphological hallmarks of prion infections. The exquisite accessibility of COCS to pharmacological manipulations was instrumental in recognizing the role of calpains in neurotoxicity, and significantly extends the collection of tools necessary for rigorously dissecting prion pathogenesis.

Long-term, prion-infected cultures of organotypic cerebellar slices to monitor neurodegeneration

Long-term, prion-infected cultures of organotypic cerebellar slices to monitor neurodegeneration

Oligodendrocyte Precursor Cell Transplantation into Organotypic Cerebellar Shiverer Slices: A Model to Study Myelination and Myelin Maintenance

Current in vitro models to investigate the consequence of oligodendrocyte-specific loss-of-function mutations on myelination are primarily limited to co-culture experiments, which do not accurately recapitulate the complex in vivo environment. Here, we describe the development of an in vitro model of myelination and myelin maintenance in which oligodendrocyte precursor cells are transplanted into organotypic cerebellar slice cultures derived from dysmyelinated shiverer mice. Compared to neuron-oligodendrocyte co-cultures, organotypic slices more closely mimic the environment in vivo, while utilizing a genetic background that allows for straight-forward identification of myelin generated by transplanted cells. We show at the ultrastructural level that the myelin generated by wild-type transplanted oligodendrocytes is compact and terminates in cytoplasmic loops that form paranodal junctions with the axon. This myelination results in the appropriate sequestering of axonal proteins into specialized domains surrounding the nodes of Ranvier. We also demonstrate the applicability of this approach for xenograft transplantation of oligodendrocyte precursor cells derived from rat or human sources. This method provides a time-efficient and cost-effective adjunct to conditional knockout mouse lines or in vivo transplantation models to study oligodendrocyte-specific loss-of-function mutations. Furthermore, the approach can be readily used to assess the effect of pharmacological manipulations on myelin, providing a tool to better understand myelination and develop effective therapeutic strategies to treat myelin-related diseases.

CNS Live Imaging Reveals a New Mechanism of Myelination: The Liquid Croissant Model (P02.065)

Objective: Even though the current model of myelination has been recognized as insufficient, CNS myelin formation has not yet been investigated in real-time with the requisite technique and resolution. Using confocal live microscopy, our study intends to provide further insight into the mechanism of myelination of the central nervous system. Background Based on electron microscopic findings more than half a century ago, the current model of myelination describes all myelin membranes to run in parallel with the longitudinal axis of the axon and to form a smooth surface, reminiscent of a rolled up carpet. However, different studies in the past demonstrated a distinct myelin morphology with an uneven myelin surface contour that challenges the established concept. Design/Methods: We therefore traced myelin growth in murine organotypic cerebellar slice cultures using high-resolution confocal live imaging, light and electron microscopy and assessed myelin morphology in young and adult mice by confocal microscopy. Results: Our data verify that the myelin surface is indeed not smooth but runs in a bidirectional, regularly spaced coil along the axon in both young and adult mice. Time-lapse imaging revealed that the growth of coiled myelin turns emerges during myelin formation. Conclusions: We therefore propose the liquid croissant model as a new concept of myelination that overcomes not only some of the incongruences of previous myelination theories, but potentially also explains the development of certain myelin pathologies observed in remyelination and axonopathies. Supported by: Swiss Multiple Sclerosis Society, Swiss National Science Foundation, 3R Research Foundation, University of Zurich, Research Commission of the Medical Faculty of the Heinrich-Heine-University Dusseldorf. Disclosure: Dr. Goebels has received personal compensation for activities with Biogen Idec, Bayer Pharmaceuticals Corporation, Merck Serono, Novartis and Teva Neuroscience as a speaker and participant on advisory boards. Dr. Sobottka has nothing to disclose. Dr. Kaech has nothing to disclose. Dr. Becher has nothing to disclose. Dr. Ziegler has nothing to disclose.

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents (3). Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells (11) are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period (4). We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.

P/Q‐type and T‐type calcium channels, but not type 3 transient receptor potential cation channels, are involved in inhibition of dendritic growth after chronic metabotropic glutamate receptor type 1 and protein kinase C activation in cerebellar Purkinje cells

The development of a neuronal dendritic tree is modulated both by signals from afferent fibers and by an intrinsic program. We have previously shown that chronic activation of either type 1 metabotropic glutamate receptors (mGluR1s) or protein kinase C (PKC) in organotypic cerebellar slice cultures of mice and rats severely inhibits the growth and development of the Purkinje cell dendritic tree. The signaling events linking receptor activation to the regulation of dendritic growth remain largely unknown. We have studied whether channels allowing the entry of Ca(2+) into Purkinje cells, in particular the type 3 transient receptor potential cation channels (TRPC3s), P/Q-type Ca(2+) channels, and T-type Ca(2+) channels, might be involved in signaling after mGluR1 or PKC stimulation. We show that the inhibition of dendritic growth seen after mGluR1 or PKC stimulation is partially rescued by pharmacological blockade of P/Q-type and T-type Ca(2+) channels, indicating that activation of these channels mediating Ca(2+) influx contributes to the inhibition of dendritic growth. In contrast, the absence of Ca(2+) -permeable TRPC3s in TRPC3-deficient mice or pharmacological blockade had no effect on mGluR1-mediated and PKC-mediated inhibition of Purkinje cell dendritic growth. Similarly, blockade of Ca(2+) influx through glutamate receptor δ2 or R-type Ca(2+) channels or inhibition of release from intracellular stores did not influence mGluR1-mediated and PKC-mediated inhibition of Purkinje cell dendritic growth. These findings suggest that both T-type and P/Q-type Ca(2+) channels, but not TRPC3 or other Ca(2+) -permeable channels, are involved in mGluR1 and PKC signaling leading to the inhibition of dendritic growth in cerebellar Purkinje cells.

S1P1 receptor subtype inhibits demyelination and regulates chemokine release in cerebellar slice cultures

Sphingosine-1-phosphate receptors (S1PRs) are drug targets for the compound FTY720, which is the first oral therapy developed for treatment of relapsing-remitting multiple sclerosis. S1PRs play a variety of functional roles in the differentiation, proliferation, survival and/or migration of neurons and glia. In this study, rat organotypic cerebellar slice cultures were used to assess whether S1PRs play a role in demyelination induced by lysolecithin (LPC). The data demonstrated that FTY720 and SEW2871 (a S1P1R-specific agonist) inhibited LPC-induced demyelination as assessed by myelin basic protein (MBP) immunofluorescence. Treatment with both drugs for 48 h also induced an increase in S1P1R expression in astrocytes. Moreover, FTY720 and SEW2871 inhibited the release of several chemokines in conditions of LPC-induced demyelination, including LIX (CXCL5), MIP-1alpha, and MIP-3alpha. Taken together, the data suggest that activation of S1P1Rs prevents LPC-induced demyelination via a mechanism involving a reduction of chemotactic chemokine release. The study supports the concept that FTY720 attenuates demyelination by not only preventing S1PR-mediated T cell migration into the CNS but also by limiting cytokine communication between cells of the immune system and the CNS.

CNS live imaging reveals a new mechanism of myelination: The liquid croissant model

The overall morphology and with it associated the formation of myelin is generally thought to be resolved. Based on electron microscopic findings more than half a century ago, the current model of myelination describes all myelin membranes to run in parallel with the longitudinal axis of the axon and to form a smooth surface, reminiscent of a rolled up carpet. However, different studies in the past demonstrated a distinct myelin morphology with an uneven myelin surface contour that challenges the established concept. Even though the current model of myelination has since been recognized as insufficient, CNS myelin formation has not yet been investigated in real-time with the requisite technique and resolution. We therefore traced myelin growth in murine organotypic cerebellar slice cultures using high-resolution confocal live imaging, light and electron microscopy and assessed myelin morphology in young and adult mice by confocal microscopy. Our data verify that the myelin surface is indeed not smooth but runs in a bidirectional, regularly spaced coil along the axon in both young and adult mice. Time-lapse imaging revealed that the growth of coiled myelin turns emerges during myelin formation. We therefore propose the "liquid croissant" model as a new concept of myelination that overcomes not only some of the incongruences of previous myelination theories, but potentially also explains the development of certain myelin pathologies observed in remyelination and axonopathies.

Foxp4 is essential in maintenance of purkinje cell dendritic arborization in the mouse cerebellum

Purkinje cells (PCs) are one of the principal neurons in the cerebellar cortex that play a central role in the coordination of fine-tuning body movement and balance. To acquire normal cerebellum function, PCs develop extensive dendritic arbors that establish synaptic connections with the parallel fibers of granule cells to form the proper neuronal circuitry. Therefore, dendritic arborization of PCs is an important developmental step to construct the mature neural network in the cerebellum. However, the genetic control of this process is not fully understood. In this study, Foxp4, a forkhead transcription factor that is expressed specifically in migrating and mature PCs of cerebellum from embryonic stages to adulthood, was knocked down by small interfering RNA (siRNA) in organotypic cerebellar slice culture. When Foxp4 expression was knocked down at postnatal day 5 (P5), no abnormalities for early dendritic remodeling of PCs were observed. However, when Foxp4 was knocked down in P10 cerebellar slices, the organization of PC dendritic arbors was highly impaired, leaving hypoplastic but non-apoptotic cell bodies. The radial alignment of Bergmann glial fibers that associated with PC dendrites was also lost. These results suggest that Foxp4 is dispensable for the early PC dendrite outgrowth, but is essential for the maintenance of PC dendritic arborization and subsequent association with Bergmann glial fibers.

Engulfment of cerebral apoptotic bodies controls the course of prion disease in a mouse strain–dependent manner

Progressive accumulation of PrPSc, a hallmark of prion diseases, occurs when conversion of PrPC into PrPSc is faster than PrPSc clearance. Engulfment of apoptotic bodies by phagocytes is mediated by Mfge8 (milk fat globule epidermal growth factor 8). In this study, we show that brain Mfge8 is primarily produced by astrocytes. Mfge8 ablation induced accelerated prion disease and reduced clearance of cerebellar apoptotic bodies in vivo, as well as excessive PrPSc accumulation and increased prion titers in prion-infected C57BL/6 × 129Sv mice and organotypic cerebellar slices derived therefrom. These phenotypes correlated with the presence of 129Sv genomic markers in hybrid mice and were not observed in inbred C57BL/6 Mfge8−/− mice, suggesting the existence of additional strain-specific genetic modifiers. Because Mfge8 receptors are expressed by microglia and depletion of microglia increases PrPSc accumulation in organotypic cerebellar slices, we conclude that engulfment of apoptotic bodies by microglia may be an important pathway of prion clearance controlled by astrocyte-borne Mfge8.

Fingolimod (FTY720) Enhances Remyelination Following Demyelination of Organotypic Cerebellar Slices

Remyelination, which occurs subsequent to demyelination, contributes to functional recovery and is mediated by oligodendrocyte progenitor cells (OPCs) that have differentiated into myelinating cells. Therapeutics that impact remyelination in the CNS could be critical determinants of long-term functional outcome in multiple sclerosis (MS). Fingolimod is a S1P receptor modulator in MS clinical trials due to systemic anti-inflammatory properties, yet may impact cells within the CNS by crossing the blood-brain barrier. Previous studies using isolated dissociated cultures indicate that neural cells express S1P receptors and respond to receptor engagement. Our objective was to assess the effects of fingolimod on myelin-related processes within a multicellular environment that maintains physiological cell-cell interactions, using organotypic cerebellar slice cultures. Fingolimod treatment had no impact on myelin under basal conditions. Fingolimod treatment subsequent to lysolecithin-induced demyelination enhanced remyelination and process extension by OPCs and mature oligodendrocytes, while increasing microglia numbers and immunoreactivity for the astrocytic marker glial fibrillary acidic protein. The number of phagocytosing microglia was not increased by fingolimod. Using S1P receptor specific agonists and antagonists, we determined that fingolimod-induced effects on remyelination and astrogliosis were mediated primarily through S1P3 and S1P5, whereas enhanced microgliosis was mediated through S1P1 and S1P5. Taken together, these data demonstrate that fingolimod modulates multiple neuroglial cell responses, resulting in enhanced remyelination in organotypic slice cultures that maintain the complex cellular interactions of the mammalian brain.

Enhancement of Magnetic Nanoparticle-Mediated Gene Transfer to Astrocytes by ‘Magnetofection‘: Effects of Static and Oscillating Fields

To assess the feasibility of using magnetic nanoparticles (MNPs) to transfect astrocytes derived for transplantation and determine if transfection efficacy can be enhanced by static and oscillating magnetic fields. Astrocytes were transfected using MNPs functionalized with a plasmid encoding a reporter protein. Transfection efficacies were compared following application of static fields and a novel, oscillating array system at a range of frequencies. The transplantation potential of transfected cells was tested in organotypic cerebellar slice cultures. Rat astrocytes can be efficiently transfected using MNPs with applied static/oscillating fields; the latter effect is frequency dependent. Transfected astrocytes could survive and differentiate following introduction into 3D neural tissue arrays. MNP vectors can safely and effectively transfect rodent astrocytes and could form the basis of a 'multifunctional nanoplatform' for neural cell transplantation.

AnkyrinG is required to maintain axo-dendritic polarity in vivo

Neurons are highly polarized cells that extend a single axon and several dendrites. Studies with cultured neurons indicate that the proximal portion of the axon, denoted as the axon initial segment (AIS), maintains neuronal polarity in vitro. The membrane-adaptor protein ankyrinG (ankG) is an essential component of the AIS. To determine the relevance of ankG for neuronal polarity in vivo, we studied mice with a cerebellum-specific ankG deficiency. Strikingly, ankG-depleted axons develop protrusions closely resembling dendritic spines. Such axonal spines are enriched with postsynaptic proteins, including ProSAP1/Shank2 and ionotropic and metabotropic glutamate receptors. In addition, immunofluorescence indicated that axonal spines are contacted by presynaptic glutamatergic boutons. For further analysis, double mutants were obtained by crossbreeding ankG(-/-) mice with L7/Purkinje cell-specific promoter 2 (PCP2) mice expressing enhanced green fluorescent protein (EGFP) in Purkinje cells (PCs). This approach allowed precise confocal microscopic mapping of EGFP-positive spiny axons and their subsequent identification at the electron microscopic level. Ultrastructurally, axonal spines contained a typical postsynaptic density and established asymmetric excitatory synapses with presynaptic boutons containing synaptic vesicles. In the shaft of spiny axons, typical ultrastructural features of the AIS, including the membrane-associated dense undercoating and cytoplasmic bundles of microtubules, were absent. Finally, using time-lapse imaging of organotypic cerebellar slice cultures, we demonstrate that nonspiny PC axons of EGFP-positive/ankG(-/-) mice acquire a spiny phenotype within a time range of only 3 days. Collectively, these findings demonstrate that axons of ankG-deficient mice acquire hallmark features of dendrites. AnkG thus is important for maintaining appropriate axo-dendritic polarity in vivo.

Reduced size of the dendritic tree does not protect Purkinje cells from excitotoxic death

Purkinje cell loss by excitotoxic damage is a typical finding in many cerebellar diseases. One important aspect of this high sensitivity of Purkinje cells to excitotoxic death might be the enormous size of their dendritic tree, with a high load of excitatory glutamate receptors. We have studied whether reduction in the size of the dendritic tree might confer resistance against excitotoxic death to Purkinje cells. We have grown Purkinje cells in organotypic cerebellar slice cultures under chronic activation of metabotropic glutamate receptors or of protein kinase C. Both treatments strongly reduced dendritic tree size. After this treatment, cells were exposed to the glutamate receptor agonist AMPA, which has a strong excitotoxic effect on Purkinje cells. We found that Purkinje cells with small dendritic trees were as sensitive to AMPA exposure as untreated control cells with large dendritic trees. Immunostaining against vesicular glutamate transporter 1 revealed that the small dendritic trees were densely covered by glutamatergic terminals. Our results indicate that the expansion of the dendritic tree and the total number of AMPA receptors per neuron do not play a major role in determining the susceptibility of Purkinje cells to excitotoxic death.

Collateral Bystander Damage by Myelin-Directed CD8 + T Cells Causes Axonal Loss

Permanent disability of patients suffering from central nervous system (CNS) inflammation such as multiple sclerosis, the most common chronic inflammatory disorder of the CNS, originates mainly from demyelination and axonal damage. Although many studies in the past focused on the role of CD4(+) T cells, several recent findings postulate the relevance of autoaggressive, cytotoxic CD8(+) T cells in the effector phase of multiple sclerosis. Yet, it remains unresolved whether axonal injury is the result of a CD8(+) T cell-targeted hit against the axon itself or the consequence of an attack against the myelin structure. To address this issue of CD8-mediated tissue damage in CNS inflammation, we performed continuous confocal imaging of autoaggressive, cytotoxic CD8(+) T cells in living organotypic cerebellar brain slices. We observed that loading brain slices with the cognate peptide antigen caused CD8-mediated damage of myelinated axons. To exclude the possibility that the cognate peptide loaded onto the brain slices was presented by axons directly, we restricted the cognate antigen expression exclusively to the cytosol of oligodendrocytes. Aside from vast myelin damage, extensive axonal bystander injury occurred. Using this model system of inflammatory CNS injury, we visualize that axonal loss can be the consequence from "collateral bystander damage" by autoaggressive, cytotoxic CD8(+) T cells, targeting their cognate antigen processed and presented by oligodendrocytes.

A molecular basis for phosphorylation-dependent SUMO conjugation by the E2 UBC9

Phosphorylation and SUMO conjugation contribute to the spatial and temporal regulation of substrates containing phosphorylation-dependent SUMO consensus motifs (PDSM). MEF2 is a transcription factor and PDSM substrate whose modification by SUMO drives postsynaptic dendritic differentiation. NMR analysis revealed that the human SUMO E2 interacted with model substrates for phosphorylated and non-phosphorylated MEF2 in similar extended conformations. Mutational and biochemical analysis identified a basic E2 surface that enhanced SUMO conjugation to phosphorylated PDSM substrates MEF2 and HSF1, but not to non-phosphorylated MEF2 or HSF1 or the non-PDSM substrate p53. Mutant Ubc9 isoforms defective in promoting SUMO conjugation to phosphorylated MEF2 in vitro and in vivo also impair postsynaptic differentiation in organotypic cerebellar slices. These data support an E2-dependent mechanism that underlies phosphorylation-dependent SUMO conjugation in pathways that range from heat shock response to nuclear hormone signaling to brain development.

Live imaging of remyelination after antibody-mediated demyelination in an ex-vivo model for immune mediated CNS damage

Mononuclear cell infiltrates, deposits of immunoglobulin and complement as well as demyelination and axonal damage are neuropathological hallmarks of Multiple Sclerosis (MS) lesions. An involvement of antibodies is further suggested by the presence of oligoclonal immunoglobulins in the cerebrospinal fluid of almost all MS patients. However, which mechanisms are most relevant for de- and remyelination and axonal loss in MS lesions is poorly understood. To characterize the regenerative abilities of demyelinated CNS tissue, we utilized murine organotypic cerebellar slice cultures expressing GFP in oligodendrocytes. The addition of a demyelinating monoclonal antibody specific for myelin oligodendrocyte glycoprotein and complement induced complete myelin destruction and oligodendrocyte loss, as demonstrated by confocal live imaging and staining for different myelin proteins. After removal of antibodies and complement we visualized the stages of remyelination, presumably originating from proliferating oligodendrocyte precursor cells and guided by morphologically intact appearing axons. Allowing for the detailed live imaging of de- and remyelination in an ex vivo situation closely resembling the three dimensional cytoarchitecture of the CNS, we provide a useful experimental system for the evaluation of new therapeutic strategies to enhance remyelination and repair in MS.

A Centrosomal Cdc20-APC Pathway Controls Dendrite Morphogenesis in Postmitotic Neurons

The ubiquitin ligase anaphase-promoting complex (APC) recruits the coactivator Cdc20 to drive mitosis in cycling cells. However, the nonmitotic functions of Cdc20-APC have remained unexplored. We report that Cdc20-APC plays an essential role in dendrite morphogenesis in postmitotic neurons. Knockdown of Cdc20 in cerebellar slices and in postnatal rats in vivo profoundly impairs the formation of granule neuron dendrite arbors in the cerebellar cortex. Remarkably, Cdc20 is enriched at the centrosome in neurons, and the centrosomal localization is critical for Cdc20-dependent dendrite development. We also find that the centrosome-associated protein histone deacetylase 6 (HDAC6) promotes the polyubiquitination of Cdc20, stimulates the activity of centrosomal Cdc20-APC, and drives the differentiation of dendrites. These findings define a postmitotic function for Cdc20-APC in the morphogenesis of dendrites in the mammalian brain. The identification of a centrosomal Cdc20-APC ubiquitin signaling pathway holds important implications for diverse biological processes, including neuronal connectivity and plasticity.

Specific JNK Inhibition by D-JNKI1 Protects Purkinje Cells from Cell Death in Lurcher Mutant Mouse

In the Lurcher mutant mouse (+/Lc), Purkinje cells (PCs) selectively die due to the mutation that converts alanine to threonine in the glutamate ionotropic receptor GRID 2, thus resulting in a constitutively leaky cation channel. This intrinsic cell death determines a target-dependent cell death of granule cells and olivary neurons and cerebellum cytoarchitecture is severely disrupted in the adult Lurcher mutant. Although the +/Lc mutant has been widely characterized, less is known about the molecules involved in +/Lc PC death. We, here, used organotypic cerebellar slice cultures from P0 mice to investigate the role of c-jun N-terminal kinase (JNK) in +/Lc PC death by using D-JNKI1 as very specific tool to inhibit its action. Our results showed that D-JNKI1 treatment increased the number of +/Lc PC at 14 DIV of 3.6-fold. Conversely, this specific JNK inhibitor cell permeable peptide did not increase PC number in +/+ treated versus untreated cultures. These results clearly indicate that JNK plays an important role in +/Lc PC mechanism of cell death.