Viewpoint: spinocerebellar ataxias as diseases of Purkinje cell dysfunction rather than Purkinje cell loss.

Spinocerebellar ataxias (SCAs) are a group of hereditary neurodegenerative diseases mostly affecting cerebellar Purkinje cells caused by a wide variety of different mutations. One subtype, SCA14, is caused by mutations of Protein Kinase C gamma (PKCγ), the dominant PKC isoform present in Purkinje cells. Mutations in the pathway in which PKCγ is active, i.e., in the regulation of calcium levels and calcium signaling in Purkinje cells, are the cause of several other variants of SCA. In SCA14, many of the observed mutations in the PKCγ gene were shown to increase the basal activity of PKCγ, raising the possibility that increased activity of PKCγ might be the cause of most forms of SCA14 and might also be involved in the pathogenesis of SCA in related subtypes. In this viewpoint and review article we will discuss the evidence for and against such a major role of PKCγ basal activity and will suggest a hypothesis of how PKCγ activity and the calcium signaling pathway may be involved in the pathogenesis of SCAs despite the different and sometimes opposing effects of mutations affecting these pathways. We will then widen the scope and propose a concept of SCA pathogenesis which is not primarily driven by cell death and loss of Purkinje cells but rather by dysfunction of Purkinje cells which are still present and alive in the cerebellum.

Transcriptome Profile of a New Mouse Model of Spinocerebellar Ataxia Type 14 Implies Changes in Cerebellar Development.

The autosomal dominant inherited spinocerebellar ataxias (SCAs) are a group of neurodegenerative disorders characterized by cerebellar atrophy and loss of Purkinje neurons. Spinocerebellar ataxia type 14 (SCA14) is a rare variant of SCAs caused by missense mutations or deletions in the PRKCG gene encoding the protein kinase C γ (PKCγ). Although mutated PKCγs are responsible for SCA14, it is still unclear exactly how mutated PKCγs are involved in SCA14 pathogenesis. Therefore, it is important to study how PKCγ signaling is altered in the cerebellum, which genes or signaling pathways are affected, and how this leads to neurological disease. In this study, we used a mouse line carrying a knock-in pseudo-substrate domain mutation in PKCγ (PKCγ-A24E) as an SCA14 model and performed RNA sequencing (RNA-seq) analysis at an early developmental timepoint (postnatal day 15) to investigate changes in the gene profile compared to wildtype mice. We analyzed both heterozygous (Het) PKCγ-A24E mice and homozygous (Homo) PKCγ-A24E mice for transcriptomic changes. The Het PKCγ-A24E mice reflects the situation observed in human SCA14 patient, while Homo PKCγ-A24E mice display stronger phenotypes with respect to Purkinje cell development and behavior. Our findings highlight an abundance of modifications affecting genes involved in developmental processes, suggesting that at least a part of the final phenotype is shaped by altered cerebellar development and is not only caused by changes in mature animals.

The Emerging Key Role of the mGluR1-PKCγ Signaling Pathway in the Pathogenesis of Spinocerebellar Ataxias: A Neurodevelopmental Viewpoint.

Spinocerebellar ataxias (SCAs) are a heterogeneous group of autosomal dominantly inherited progressive disorders with degeneration and dysfunction of the cerebellum. Although different subtypes of SCAs are classified according to the disease-associated causative genes, the clinical syndrome of the ataxia is shared, pointing towards a possible convergent pathogenic pathway among SCAs. In this review, we summarize the role of SCA-associated gene function during cerebellar Purkinje cell development and discuss the relationship between SCA pathogenesis and neurodevelopment. We will summarize recent studies on molecules involved in SCA pathogenesis and will focus on the mGluR1-PKCγ signaling pathway evaluating the possibility that this might be a common pathway which contributes to these diseases.

CRISPR-Cas13-Mediated Knockdown of Regulator of G-Protein Signaling 8 (RGS8) Does Not Affect Purkinje Cell Dendritic Development.

CRISPR-Cas13 technology is rapidly evolving as it is a very specific tool for RNA editing and interference. Since there are no significant off-target effects via the Cas13-mediated method, it is a promising tool for studying gene function in differentiating neurons. In this study, we designed two crRNA targeting regulator of G-protein signaling 8 (RGS8), which is a signaling molecule associated with spinocerebellar ataxias. Using CRISPR-Cas13 technology, we found that both of crRNAs could specifically achieve RGS8 knockdown. By observing and comparing the dendritic growth of Purkinje cells, we found that CRISPR-Cas13-mediated RGS8 knockdown did not significantly affect Purkinje cell dendritic development. We further tested the role of RGS8 by classical RNAi. Again, the results of the RNAi-mediated RGS8 knockdown showed that reduced RGS8 expression did not significantly affect the dendritic growth of Purkinje cells. This is the first example of CRISPR-Cas13-mediated gene function study in Purkinje cells and establishes CRISPR-Cas13-mediated knockdown as a reliable method for studying gene function in primary neurons.

Serine/threonine kinase 17b (STK17B) signalling regulates Purkinje cell dendritic development and is altered in multiple spinocerebellar ataxias.

Serine/threonine kinase 17b (STK17B, also known as DRAK2) is known to be a downstream effector of protein kinase C (PKC) in the immune system, in particular T lymphocytes. PKC activity also plays a critical role for dendritic development and synaptic maturation and plasticity in cerebellar Purkinje cells. We present evidence that STK17B is strongly expressed in mouse cerebellar Purkinje cells starting in the early postnatal period and remaining highly expressed throughout adult stages and that STK17B is a target of PKC phosphorylation in the cerebellum. STK17B overexpression potentiates the morphological changes of Purkinje cells seen after PKC activation, suggesting that it is a downstream effector of PKC. A phosphorylation mimetic STK17B variant induced a marked reduction of Purkinje cell dendritic tree size, whereas the inhibition of STK17B with the novel compound 16 (Cpd16) could partially rescue the morphological changes of the Purkinje cell dendritic tree after PKC activation. These findings show that STK17B signalling is an important downstream effector of PKC activation in Purkinje cells. Furthermore, STK17B was identified as a molecule being transcriptionally downregulated in mouse models of SCA1, SCA7, SCA14 and SCA41. The reduced expression of STK17B in these mouse models might protect Purkinje cell dendrites from the negative effects of overactivated PKC signalling. Our findings provide new insights in the role of STK17B for Purkinje cell dendritic development and the pathology of SCAs.

The Bacterial Enzyme RfxCas13d Is Less Neurotoxic Than PspCas13b and Could Be a Promising RNA Editing and Interference Tool in the Nervous System.

RNA therapies using RNA editing and interference are currently being developed for neurological diseases. The CRISPR-Cas13 system, based on bacterial enzymes, holds great promise for developing efficient tools for RNA therapies. However, neurotoxic activity has been reported for Cas13a, and recent studies have reported toxic effects of PspCas13b and RfxCas13d during zebrafish and Drosophila embryonic development. It is important to investigate the safety of these bacterial enzymes in the context of the nervous system and neuronal development. In this study, we used mouse cerebellar Purkinje cells as a complex neuron type to test for the potential neurotoxic actions of RfxCas13d and PspCas13b. We found that PspCas13b significantly impeded the dendritic development of cultured Purkinje cells, similar to the neurotoxic action of Cas13a. In contrast, RfxCas13d did not exhibit a significant inhibition of dendritic development. A similar trend was found for axonal outgrowth. These results suggest varying neurotoxic properties for different Cas13 ortholog enzymes. We call for more studies to investigate, and possibly mitigate, the neurotoxicity of Cas13 proteins in order to improve the safety of the CRISPR-Cas13 system for RNA therapies.

The Bacterial Enzyme Cas13 Interferes with Neurite Outgrowth from Cultured Cortical Neurons.

The CRISPR-Cas13 system based on a bacterial enzyme has been explored as a powerful new method for RNA manipulation. Due to the high efficiency and specificity of RNA editing/interference achieved by this system, it is currently being developed as a new therapeutic tool for the treatment of neurological and other diseases. However, the safety of this new generation of RNA therapies is still unclear. In this study, we constructed a vector expressing CRISPR-Cas13 under a constitutive neuron-specific promoter. CRISPR-Cas13 from Leptotrichia wadei was expressed in primary cultures of mouse cortical neurons. We found that the presence of CRISPR-Cas13 impedes the development of cultured neurons. These results show a neurotoxic action of Cas13 and call for more studies to test for and possibly mitigate the toxic effects of Cas13 enzymes in order to improve CRISPR-Cas13-based tools for RNA targeting.

Conditional gene silencing via a CRISPR system in cerebellar Purkinje cells.

• Specific gene knockdown in cerebellar Purkinje cells is a major challenge because Purkinje cells only make up a small fraction of the cerebellum and off-target effects of shRNA occur. • Little is known about the application of CRISPR-Cas13 and the resulting protein expression levels in Purkinje cells, a type of developing postmitotic neuron. • We explored the possibility of a Cas13-mediated conditional knockdown system for cerebellar Purkinje cells. This system can achieve a suppression of the target proteins restricted to Purkinje cells in mixed cerebellar cultures.

A New Mouse Model Related to SCA14 Carrying a Pseudosubstrate Domain Mutation in PKCγ Shows Perturbed Purkinje Cell Maturation and Ataxic Motor Behavior.

Spinocerebellar ataxias (SCAs) are diseases characterized by cerebellar atrophy and loss of Purkinje neurons caused by mutations in diverse genes. In SCA14, the disease is caused by point mutations or small deletions in protein kinase C γ (PKCγ), a crucial signaling protein in Purkinje cells. It is still unclear whether increased or decreased PKCγ activity may be involved in the SCA14 pathogenesis. In this study, we present a new knock-in mouse model related to SCA14 with a point mutation in the pseudosubstrate domain, PKCγ-A24E, known to induce a constitutive PKCγ activation. In this protein conformation, the kinase domain of PKCγ is activated, but at the same time the protein is subject to dephosphorylation and protein degradation. As a result, we find a dramatic reduction of PKCγ protein expression in PKCγ-A24E mice of either sex. Despite this reduction, there is clear evidence for an increased PKC activity in Purkinje cells from PKCγ-A24E mice. Purkinje cells derived from PKCγ-A24E have short thickened dendrites typical for PKC activation. These mice also develop a marked ataxia and signs of Purkinje cell dysfunction making them an interesting new mouse model related to SCA. Recently, a similar mutation in a human patient was discovered and found to be associated with overt SCA14. RNA profiling of PKCγ-A24E mice showed a dysregulation of related signaling pathways, such as mGluR1 or mTOR. Our results show that the induction of PKCγ activation in Purkinje cells results in the SCA-like phenotype indicating PKC activation as one pathogenetic avenue leading to a SCA.SIGNIFICANCE STATEMENT Spinocerebellar ataxias (SCAs) are hereditary diseases affecting cerebellar Purkinje cells and are a one of neurodegenerative diseases. While mutation in several genes have been identified as causing SCAs, it is unclear how these mutations cause the disease phenotype. Mutations in PKCγ cause one subtype of SCAs, SCA14. In this study, we have generated a knock-in mouse with a mutation in the pseudosubstrate domain of PKCγ, which keeps PKCγ in the constitutive active open conformation. We show that this mutation leading to a constant activation of PKCγ results in a SCA-like phenotype in these mice. Our findings establish the constant activation of PKC signaling as one pathogenetic avenue leading to an SCA phenotype and a mechanism causing a neurodegenerative disease.

PKCγ-Mediated Phosphorylation of CRMP2 Regulates Dendritic Outgrowth in Cerebellar Purkinje Cells.

The signalling protein PKCγ is a major regulator of Purkinje cell development and synaptic function. We have shown previously that increased PKCγ activity impairs dendritic development of cerebellar Purkinje cells. Mutations in the protein kinase Cγ gene (PRKCG) cause spinocerebellar ataxia type 14 (SCA14). In a transgenic mouse model of SCA14 expressing the human S361G mutation, Purkinje cell dendritic development is impaired in cerebellar slice cultures similar to pharmacological activation of PKC. The mechanisms of PKCγ-driven inhibition of dendritic growth are still unclear. Using immunoprecipitation-coupled mass spectrometry analysis, we have identified collapsin response mediator protein 2 (CRMP2) as a protein interacting with constitutive active PKCγ(S361G) and confirmed the interaction with the Duolink™ proximity ligation assay. We show that in cerebellar slice cultures from PKCγ(S361G)-mice, phosphorylation of CRMP2 at the known PKC target site Thr555 is increased in Purkinje cells confirming phosphorylation of CRMP2 by PKCγ. miRNA-mediated CRMP2 knockdown decreased Purkinje cell dendritic outgrowth in dissociated cerebellar cultures as did the transfection of CRMP2 mutants with a modified Thr555 site. In contrast, dendritic development was normal after wild-type CRMP2 overexpression. In a novel knock-in mouse expressing only the phospho-defective T555A-mutant CRMP2, Purkinje cell dendritic development was reduced in dissociated cultures. This reduction could be rescued by transfecting wild-type CRMP2 but only partially by the phospho-mimetic T555D-mutant. Our findings establish CRMP2 as an important target of PKCγ phosphorylation in Purkinje cells mediating its control of dendritic development. Dynamic regulation of CRMP2 phosphorylation via PKCγ is required for its correct function.

Modulation of Increased mGluR1 Signaling by RGS8 Protects Purkinje Cells From Dendritic Reduction and Could Be a Common Mechanism in Diverse Forms of Spinocerebellar Ataxia.

Spinocerebellar ataxias (SCAs) are a group of hereditary neurodegenerative diseases which are caused by diverse genetic mutations in a variety of different genes. We have identified RGS8, a regulator of G-protein signaling, as one of the genes which are dysregulated in different mouse models of SCA (e.g., SCA1, SCA2, SCA7, and SCA14). In the moment, little is known about the role of RGS8 for pathogenesis of spinocerebellar ataxia. We have studied the expression of RGS8 in the cerebellum in more detail and show that it is specifically expressed in mouse cerebellar Purkinje cells. In a mouse model of SCA14 with increased PKCγ activity, RGS8 expression was also increased. RGS8 overexpression could partially counteract the negative effects of DHPG-induced mGluR1 signaling for the expansion of Purkinje cell dendrites. Our results suggest that the increased expression of RGS8 is an important mediator of mGluR1 pathway dysregulation in Purkinje cells. These findings provide new insights in the role of RGS8 and mGluR1 signaling in Purkinje cells and for the pathology of SCAs.

The RNA-Binding Protein RBM3 Promotes Neural Stem Cell (NSC) Proliferation Under Hypoxia.

Neural stem cells (NSCs) reside physiologically in a hypoxic niche to maintain self-renewal and multipotency. Whereas mild hypoxia is known to promote NSC proliferation, severe hypoxia in pathological conditions exerts the reverse effect. The multi-functional RNA-binding protein RBM3 is abundant in NSCs and can be regulated by hypoxic exposure. Although RBM3 has been shown to accelerate cell growth in many cell types, whether and how it affects NSC proliferation in hypoxic environment remains largely unknown. In this study, we tested how RBM3 regulates cell proliferation under hypoxia in C17.2 mouse NSC cell line and in primary mouse NSCs from both the forebrain of postnatal day 0 (P0) mice and the subgranular zone (SGZ) of adult mice. Our results demonstrated that RBM3 expression was highly sensitive to hypoxia, and NSCs were arrested in G0/G1 phase by 5, 2.5, and 1% O2 treatment. When we overexpressed RBM3, hypoxia-induced cell cycle arrest in G0/G1 phase was relieved and more cell transit into S phase was observed. Furthermore, cell viability under hypoxia was also increased by RBM3. In contrast, in RBM3-depleted primary NSCs, less BrdU-incorporated cells were detected, indicating exacerbated cell cycle arrest in G1 to S phase transition. Instead, overexpressed RBM3 significantly increased proliferation ratio in primary NSCs. Our findings indicate RBM3 as a potential target to maintain the proliferation capacity of NSCs under hypoxia, which can be important in NSC-based therapies of acute brain injury and chronic neurodegenerative diseases.

RBM3 promotes neurogenesis in a niche-dependent manner via IMP2-IGF2 signaling pathway after hypoxic-ischemic brain injury.

Hypoxic ischemia (HI) is an acute brain threat across all age groups. Therapeutic hypothermia ameliorates resulting injury in neonates but its side effects prevent routine use in adults. Hypothermia up-regulates a small protein subset that includes RNA-binding motif protein 3 (RBM3), which is neuroprotective under stressful conditions. Here we show how RBM3 stimulates neuronal differentiation and inhibits HI-induced apoptosis in the two areas of persistent adult neurogenesis, the subventricular zone (SVZ) and the subgranular zone (SGZ), while promoting neural stem/progenitor cell (NSPC) proliferation after HI injury only in the SGZ. RBM3 interacts with IGF2 mRNA binding protein 2 (IMP2), elevates its expression and thereby stimulates IGF2 release in SGZ but not SVZ-NSPCs. In summary, we describe niche-dependent regulation of neurogenesis after adult HI injury via the novel RBM3-IMP2-IGF2 signaling pathway.

Reduced Purkinje cell size is compatible with near normal morphology and function of the cerebellar cortex in a mouse model of spinocerebellar ataxia.

Spinocerebellar ataxia type 14 (SCA14) is a dominantly inherited neurodegenerative disease caused by diverse mutations in the Protein Kinase C gamma (PKCγ) gene which is one of the crucial signaling molecules of Purkinje cells. We have previously created a mouse model of SCA14 by transgenic expression of a mutated PKCγ gene causing SCA14 with a mutation in the catalytic domain. Purkinje cells from the mutated mice have a strong reduction of their dendritic tree in organotypic slice cultures typical for increased PKC activity. There was no overt degeneration of Purkinje cells in vivo and the cerebellum appeared morphologically normal with the exception of lobule 7 where abnormal Purkinje cells were present. Besides from mild motor deficits the mice have no major phenotype. We have now done a more extensive study of cerebellar morphology in these mice and show by rapid Golgi staining that there is a marked reduction of Purkinje cell dendritic tree size throughout the cerebellum. Despite this reduction in dendritic tree size, climbing fiber innervation of Purkinje cells as visualized by immunostaining for the vesicular glutamate transporter 2 (vGlut2) appeared normal in most parts of the cerebellum. The same was true for the expression of the activity and plasticity markers pS6, c-Fos and Arc. These finding suggest that the cerebellar cortex in the transgenic mice is functioning fairly normal and that the reduction of dendritic tree size and the increased PKC activity can be compensated in most Purkinje cells. Around cerebellar lobule 7 there was high transgene expression from the L7 promotor and Purkinje cells showed abnormal morphologies. Climbing fiber innervation as well as the expression of the activity and plasticity markers was strongly disturbed in this area. Our results show that there is substantial potential for functional compensation in the cerebellar cortex. In lobule 7, an area with high transgene expression, compensation failed resulting in Purkinje cell degeneration and dysfunction.

Calcium Signaling, PKC Gamma, IP3R1 and CAR8 Link Spinocerebellar Ataxias and Purkinje Cell Dendritic Development.

BACKGROUND: Spinocerebellar ataxias (SCAs) are a group of cerebellar diseases characterized by progressive ataxia and cerebellar atrophy. Several forms of SCAs are caused by missense mutations or deletions in genes related to calcium signaling in Purkinje cells. Among them, spinocerebellar ataxia type 14 (SCA14) is caused by missense mutations in PRKCG gene which encodes protein kinase C gamma (PKCγ). It is remarkable that in several cases in which SCA is caused by point mutations in an individual gene, the affected genes are involved in the PKCγ signaling pathway and calcium signaling which is not only crucial for proper Purkinje cell function but is also involved in the control of Purkinje cell dendritic development. In this review, we will focus on the PKCγ signaling related genes and calcium signaling related genes then discuss their role for both Purkinje cell dendritic development and cerebellar ataxia. METHODS: Research related to SCAs and Purkinje cell dendritic development is reviewed. RESULTS: PKCγ dysregulation causes abnormal Purkinje cell dendritic development and SCA14. Carbonic anhydrase related protein 8 (Car8) encoding CAR8 and Itpr1 encoding IP3R1were identified as upregulated genes in one of SCA14 mouse model. IP3R1, CAR8 and PKCγ proteins are strongly and specifically expressed in Purkinje cells. The common function among them is that they are involved in the regulation of calcium homeostasis in Purkinje cells and their dysfunction causes ataxia in mouse and human. Furthermore, disruption of intracellular calcium homeostasis caused by mutations in some calcium channels in Purkinje cells links to abnormal Purkinje cell dendritic development and the pathogenesis of several SCAs. CONCLUSION: Once PKCγ signaling related genes and calcium signaling related genes are disturbed, the normal dendritic development of Purkinje cells is impaired as well as the integration of signals from other neurons, resulting in abnormal development, cerebellar dysfunction and eventually Purkinje cell loss.

Increased biological activity of protein Kinase C gamma is not required in Spinocerebellar ataxia 14.

Spinocerebellar ataxia (SCA) is an autosomal dominant neurodegenerative disorder characterized by slowly progressive cerebellar dysfunction. Currently, 42 SCA types are known, some of which are caused by CAG repeat expansions, but others are caused by point mutations or deletions. Spinocerebellar ataxia type 14 (SCA14) is caused by missense mutations or deletions in the PRKCG gene, coding for protein kinase C gamma (PKCγ). It is still not well understood how these mutations eventually cause Purkinje cell dysfunction and death. Because PKCγ is a well characterized signaling protein highly expressed in Purkinje cells SCA14 offers the chance to better understand the pathogenesis of Purkinje cell dysfunction and death. Altered biological activity of PKCγ would be the simplest explanation for the disease phenotype. There are indeed indications that the enzymatic activity of mutated PKCγ proteins could be changed. Many mutations found in SCA14 families are located in the regulatory C1B and C1A domain, while a few mutations are also found in the C2 and in the catalytic C3 and C4 domains. For many of these mutations an increased enzymatic activity could be demonstrated in cell-based assays, but it remains unclear whether there is indeed an altered biological activity of the mutated PKCγ proteins within living Purkinje cells. In this study we used the dendritic morphology of developing Purkinje cells to detect increased biological activity of PKCγ after expression of different mutated PKCγ proteins. Our results indicate that two out of three known mutations in the catalytic domain of PKCγ did indeed show increased biological activity. On the other hand, none of the five tested mutations located in the regulatory C1 or the C2 domain showed an increased biological activity. Our findings indicate that SCA14 mutations located in different domains of the PRKCG gene cause SCA14 by different mechanisms and that an increased constitutive activity of PKCγ may be one, but cannot be the only mechanism to cause disease in SCA14.

Chronic pharmacological blockade of the Na+ /Ca2+ exchanger modulates the growth and development of the Purkinje cell dendritic arbor in mouse cerebellar slice cultures.

The Na+ /Ca2+ exchanger (NCX) is a bidirectional plasma membrane antiporter involved in Ca2+ homeostasis in eukaryotes. NCX has three isoforms, NCX1-3, and all of them are expressed in the cerebellum. Immunostaining on cerebellar slice cultures indicates that NCX is widely expressed in the cerebellum, including expression in Purkinje cells. The pharmacological blockade of the forward mode of NCX (Ca2+ efflux mode) by bepridil moderately inhibited growth and development of Purkinje cell dendritic arbor in cerebellar slice cultures. However, the blockade of the reverse mode (Ca2+ influx mode) by KB-R7943 severely reduced the dendritic arbor and induced a morphological change with thickened distal dendrites. The effect of KB-R7943 on dendritic growth was unrelated to the activity of voltage-gated calcium channels and was also apparent in the absence of bioelectrical activity indicating that it was mediated by NCX expressed in Purkinje cells. We have used additional NCX inhibitors including CB-DMB, ORM-10103, SEA0400, YM-244769, and SN-6 which have higher specificity for NCX isoforms and target either the forward, reverse, or both modes. These inhibitors caused a strong dendritic reduction similar to that seen with KB-R7943, but did not elicit thickening of distal dendrites. Our findings indicate that disturbance of the NCX-dependent calcium transport in Purkinje cells induces a reduction of dendritic arbor, which is presumably caused by changes in the calcium handling, and underline the importance of the calcium equilibrium for the dendritic development in cerebellar Purkinje cells.

Amikacin Inhibits miR-497 Maturation and Exerts Post-ischemic Neuroprotection.

MicroRNAs (miRNAs) are a group of small non-coding RNAs that regulate numerous signaling pathways involved in cerebral ischemia reperfusion injury. Recent finding demonstrated that miR-497 promotes ischemic neuronal death by negatively regulating anti-apoptotic proteins and therefore serves as a promising therapeutic target for cerebral ischemic injury. In this study, we present a systematic computational approach that includes 3D modeling, docking-based virtual screening, and molecular dynamics simulation to identify small-molecule inhibitors of pre-miR-497 maturation. The top hit, aminoglycosidic antibiotic, amikacin, formed a stable complex with pre-miR-497. Later, the protective efficacy of amikacin was evaluated against oxygen-glucose deprivation (OGD) and reoxygenation-induced neuronal cell death in SH-SY5Y cells and mouse organotypic hippocampal slice cultures. To confirm the inhibitory potential of amikacin on miR-497 maturation, quantitative real-time PCR was performed to check the expression of bcl-2, one of the primary anti-apoptotic targets of miR-497. Additionally, the expression level of mature miR-497 was quantified using TaqMan® MiRNA Assay Kit. Amikacin treatment effectively reduced OGD-induced cell death compared to control groups both in vitro and organotypic hippocampal slice cultures. Further, amikacin effectively increased the expression of bcl-2 in SH-SY5Y cells subjected to OGD. Interestingly, SH-SY5Y cells treated with amikacin displayed decreased expression of miR-497, probably due to inhibition of pre-miRic form. Our study provides strong evidence that amikacin inhibits miR-497 maturation and promotes ischemic neuronal survival by upregulating anti-apoptotic protein, bcl-2. Future studies directed at evaluating the neuroprotective efficacy and mechanism of amikacin animal models may lead to new therapeutic opportunities for preventing neuronal death after stroke.

Carbonic Anhydrase 8 Expression in Purkinje Cells Is Controlled by PKCγ Activity and Regulates Purkinje Cell Dendritic Growth.

Purkinje cell dendritic development is severely compromised after chronic activation of protein kinase C (PKC). In a recent transgenic mouse model of spinocerebellar ataxia 14, the ser361-to-gly (S361G) mutation of the protein kinase C gamma (PKCγ) gene was expressed in Purkinje cells. Purkinje cells from these mutant mice in organotypic slice cultures have the same stunted dendritic tree as Purkinje cells after pharmacological activation of PKC. Because the transgene is exclusively present in Purkinje cells, cerebellar tissue from these mice is an attractive starting material for searching genes which might be interacting with PKCγ in Purkinje cells for inducing the stunted dendritic growth. We have performed a microarray analysis and identified several candidate genes with an increased messenger RNA (mRNA) expression in the PKCγ-S361G transgenic Purkinje cells. Out of these candidates, we have further studied carbonic anhydrase 8 (CA8). We show here that CA8 mRNA and protein expression is strongly induced in PKCγ-S361G transgenic Purkinje cells. Overexpression of CA8 in Purkinje cells in dissociated cultures strongly inhibited Purkinje cell dendritic development and produced a dendritic phenotype similar to PKCγ-S361G. There was no evidence for a direct binding of CA8 to either PKCγ or the type 1 IP3 receptor. Knockdown of CA8 with miRNA did not alter Purkinje cell dendritic development and did not protect Purkinje cells in dissociated cultures from the stunted dendritic growth induced by PKCγ-S361G or by PKC activation. Our results indicate that CA8 is a novel important regulator of Purkinje cell dendritic development and that its expression is controlled by PKCγ activity.