Search

The cerebellum, the site where protein kinase C (PKC) was first discovered, contains the highest amount of PKC in the central nervous system, with PKCγ being the major isoform. Systemic PKCγ-knockout (KO) mice showed impaired motor coordination and deficient pruning of surplus climbing fibers (CFs) from developing cerebellar Purkinje cells (PCs). However, the physiological significance of PKCγ in the mature cerebellum and the cause of motor incoordination remain unknown. Using adeno-associated virus vectors targeting PCs, we showed that impaired motor coordination was restored by re-expression of PKCγ in mature PKCγ-KO mouse PCs in a kinase activity-dependent manner, while normal motor coordination in mature Prkcg fl/fl mice was impaired by the Cre-dependent removal of PKCγ from PCs. Notably, the rescue or removal of PKCγ from mature PKCγ-KO or Prkcg fl/fl mice, respectively, did not affect the CF innervation profile of PCs, suggesting the presence of a mechanism distinct from multiple CF innervation of PCs for the motor defects in PKCγ-deficient mice. We found marked potentiation of Ca 2+ -activated large-conductance K + (BK) channel currents in PKCγ-deficient mice, as compared to wild-type mice, which decreased the membrane resistance, resulting in attenuation of the electrical signal during the propagation and significant alterations of the complex spike waveform. These changes in PKCγ-deficient mice were restored by the rescue of PKCγ or pharmacological suppression of BK channels. Our results suggest that PKCγ is a critical regulator that negatively modulates BK currents in PCs, which significantly influences PC output from the cerebellar cortex and, eventually, motor coordination., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

Spinocerebellar ataxias (SCAs) are a group of genetic diseases characterized by progressive ataxia and neurodegeneration, often in cerebellar Purkinje neurons. A SCA1 mouse model, Pcp2-ATXN1[30Q]D776, has severe ataxia in absence of progressive Purkinje neuron degeneration and death. Previous RNA-seq analyses identify cerebellar upregulation of the peptide hormone cholecystokinin (Cck) in Pcp2-ATXN1[30Q]D776 mice. Importantly, absence of Cck1 receptor (Cck1R) in Pcp2-ATXN1[30Q]D776 mice confers a progressive disease with Purkinje neuron death. Administration of a Cck1R agonist, A71623, to Pcp2-ATXN1[30Q]D776;Cck -/- and Pcp2-AXTN1[82Q] mice dampens Purkinje neuron pathology and associated deficits in motor performance. In addition, A71623 administration improves motor performance of Pcp2-ATXN2[127Q] SCA2 mice. Moreover, the Cck1R agonist A71623 corrects mTORC1 signaling and improves expression of calbindin in cerebella of AXTN1[82Q] and ATXN2[127Q] mice. These results indicate that manipulation of the Cck-Cck1R pathway is a potential therapeutic target for treatment of diseases involving Purkinje neuron degeneration., Competing Interests: Declaration of interests E.A.L.W. and H.T.O. declare patent US 10,973,812 B2 (issued April 13, 2021) as relevant to the work in this paper., (Copyright © 2021. Published by Elsevier Inc.)

The immunohistochemical pattern of kynurenine aminotransferase-2 (KAT-2) - the key role enzyme in the production of neuroactive and neuroprotective kynurenic acid (KYNA) - was studied in the cerebellum of mice. It is known from literature that KAT-2 is localized mainly in astrocytes in different parts of the cerebrum. Kynurenine aminotransferase (KAT) activity in the cerebellum is relatively low and alternative production routes for KYNA have been described there. Therefore we examined the immunohistochemical pattern of KAT-2 in this part of the brain. Surprisingly, the cellular localization of KAT-2 in mice was proven to be unique; it localized characteristically in Purkinje cells and in some other types of neurons (not identified) but was not found in astrocytes nor microglia. The exclusive neuronal, but not glial localization of KAT-2 in the cerebellum is novel and may be related to its low activity and to the alternative pathways for KYNA production that have been described., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Studies of neuroglial interaction largely depend on cell-specific gene knockout (KO) experiments using Cre recombinase. However, genes known as glial-specific genes have recently been reported to be expressed in neuroglial stem cells, leading to the possibility that a glia-specific Cre driver results in unwanted gene deletion in neurons, which may affect sound interpretation. 2',3'-Cyclic nucleotide 3'-phosphodiesterase (CNP) is generally considered to be an oligodendrocyte (OL) marker. Accordingly, Cnp promoter-controlled Cre recombinase has been used to create OL-specific gene targeting mice. However, in this study, using Rosa26-tdTomato-reporter/Cnp-Cre mice, we found that many forebrain neurons and cerebellar Purkinje neurons belong to the lineages of Cnp-expressing neuroglial stem cells. To answer whether gene targeting by Cnp-Cre can induce neuron-autonomous defects, we conditionally deleted an essential autophagy gene, Atg7, in Cnp-Cre mice. The Cnp-Cre-mediated Atg7 KO mice showed extensive p62 inclusion in neurons, including cerebellar Purkinje neurons with extensive neurodegeneration. Furthermore, neuronal areas showing p62 inclusion in Cnp-Cre-mediated Atg7 KO mice overlapped with the neuronal lineage of Cnp-expressing neuroglial stem cells. Moreover, Cnp-Cre-mediated Atg7-KO mice did not develop critical defects in myelination. Our results demonstrate that a large population of central neurons are derived from Cnp-expressing neuroglial stem cells; thus, conditional gene targeting using the Cnp promoter, which is known to be OL-specific, can induce neuron-autonomous phenotypes., (© 2020 Federation of American Societies for Experimental Biology.)

Background: Polyamine catabolism plays a key role in maintaining intracellular polyamine pools, yet its physiological significance is largely unexplored. Here, we report that the disruption of polyamine catabolism leads to severe cerebellar damage and ataxia, demonstrating the fundamental role of polyamine catabolism in the maintenance of cerebellar function and integrity., Methods: Mice with simultaneous deletion of the two principal polyamine catabolic enzymes, spermine oxidase and spermidine/spermine N 1 -acetyltransferase (Smox/Sat1-dKO), were generated by the crossbreeding of Smox-KO (Smox -/- ) and Sat1-KO (Sat1 -/- ) animals. Development and progression of tissue injury was monitored using imaging, behavioral, and molecular analyses., Results: Smox/Sat1-dKO mice are normal at birth, but develop progressive cerebellar damage and ataxia. The cerebellar injury in Smox/Sat1-dKO mice is associated with Purkinje cell loss and gliosis, leading to neuroinflammation and white matter demyelination during the latter stages of the injury. The onset of tissue damage in Smox/Sat1-dKO mice is not solely dependent on changes in polyamine levels as cerebellar injury was highly selective. RNA-seq analysis and confirmatory studies revealed clear decreases in the expression of Purkinje cell-associated proteins and significant increases in the expression of transglutaminases and markers of neurodegenerative microgliosis and astrocytosis. Further, the α-Synuclein expression, aggregation, and polyamination levels were significantly increased in the cerebellum of Smox/Sat1-dKO mice. Finally, there were clear roles of transglutaminase-2 (TGM2) in the cerebellar pathologies manifest in Smox/Sat1-dKO mice, as pharmacological inhibition of transglutaminases reduced the severity of ataxia and cerebellar injury in Smox/Sat1-dKO mice., Conclusions: These results indicate that the disruption of polyamine catabolism, via coordinated alterations in tissue polyamine levels, elevated transglutaminase activity and increased expression, polyamination, and aggregation of α-Synuclein, leads to severe cerebellar damage and ataxia. These studies indicate that polyamine catabolism is necessary to Purkinje cell survival, and for sustaining the functional integrity of the cerebellum.

While in birds and mammals the cerebellum is a highly convoluted structure that consists of numerous transverse lobules, in most amphibians and reptiles it consists of only a single unfolded sheet. Orthogonal to the lobules, the cerebellum is comprised of sagittal zones that are revealed in the pattern of afferent inputs, the projection patterns of Purkinje cells, and Purkinje cell response properties, among other features. The expression of several molecular markers, such as aldolase C, is also parasagittally organized. Aldolase C, also known as zebrin II (ZII), is a glycolytic enzyme expressed in the cerebellar Purkinje cells of the vertebrate cerebellum. In birds, mammals, and some lizards (Ctenophoresspp.), ZII is expressed in a heterogenous fashion of alternating sagittal bands of high (ZII+) and low (ZII-) expression Purkinje cells. In contrast, turtles and snakes express ZII homogenously (ZII+) in their cerebella, but the pattern in crocodilians is unknown. Here, we examined the expression of ZII in two crocodilian species (Crocodylus niloticus and Alligator mississippiensis) to help determine the evolutionary origin of striped ZII expression in vertebrates. We expected crocodilians to express ZII in a striped (ZII+/ZII-) manner because of their close phylogenetic relationship to birds and their larger and more folded cerebellum compared to that of snakes and turtles. Contrary to our prediction, all Purkinje cells in the crocodilian cerebellum had a generally homogenous expression of ZII (ZII+) rather than clear ZII+/- stripes. Our results suggest that either ZII stripes were lost in three groups (snakes, turtles, and crocodilians) or ZII stripes evolved independently three times (lizards, birds, and mammals)., (© 2020 S. Karger AG, Basel.)

Spinocerebellar ataxias (SCA) are a genetically heterogeneous family of cerebellar neurodegenerative diseases characterized by abnormal firing of Purkinje neurons and degeneration. We recently demonstrated the slowed firing rates seen in several SCAs share a common etiology of hyper-activation of the Src family of non-receptor tyrosine kinases (SFKs). However, the lack of clinically available neuroactive SFK inhibitors lead us to investigate alternative mechanisms to modulate SFK activity. Previous studies demonstrate that SFK activity can be enhanced by the removal of inhibitory phospho-marks by receptor-protein-tyrosine phosphatases (RPTPs). In this Extra View we show that MTSS1 inhibits SFK activity through the binding and inhibition of a subset of the RPTP family members, and lowering RPTP activity in cerebellar slices with peptide inhibitors increases the suppressed Purkinje neuron basal firing rates seen in two different SCA models. Together these results identify RPTPs as novel effectors of Purkinje neuron basal firing, extending the MTSS1/SFK regulatory circuit we previously described and expanding the therapeutic targets for SCA patients.

Mitochondrial dysfunction causes neurodegeneration but whether impairment of mitochondrial homeostasis in astrocytes contributes to this pathological process remains largely unknown. The m-AAA protease exerts quality control and regulatory functions crucial for mitochondrial homeostasis. AFG3L2, which encodes one of the subunits of the m-AAA protease, is mutated in spinocerebellar ataxia SCA28 and in infantile syndromes characterized by spastic-ataxia, epilepsy and premature death. Here, we investigate the role of Afg3l2 and its redundant homologue Afg3l1 in the Bergmann glia (BG), radial astrocytes of the cerebellum that have functional connections with Purkinje cells (PC) and regulate glutamate homeostasis. We show that astrocyte-specific deletion of Afg3l2 in the mouse leads to late-onset motor impairment and to degeneration of BG, which display aberrant morphology, altered expression of the glutamate transporter EAAT2, and a reactive inflammatory signature. The neurological and glial phenotypes are drastically exacerbated when astrocytes lack both Afg31l and Afg3l2, and therefore, are totally depleted of the m-AAA protease. Moreover, mitochondrial stress responses and necroptotic markers are induced in the cerebellum. In both mouse models, targeted BG show a fragmented mitochondrial network and loss of mitochondrial cristae, but no signs of respiratory dysfunction. Importantly, astrocyte-specific deficiency of Afg3l1 and Afg3l2 triggers secondary morphological degeneration and electrophysiological changes in PCs, thus demonstrating a non-cell-autonomous role of glia in neurodegeneration. We propose that astrocyte dysfunction amplifies both neuroinflammation and glutamate excitotoxicity in patients carrying mutations in AFG3L2, leading to a vicious circle that contributes to neuronal death., (© 2019 The Authors. Glia published by Wiley Periodicals, Inc.)

Objectives: Mechanisms underlying Purkinje cell (PC) death, which leads to many diseases in humans, are still poorly elucidated. Progressive PC degeneration occurs in shaker mutant rat due to an X-linked recessive mutation leading to gait ataxia and total-body tremors. Chemoablation of the inferior olive (IO) and olivocerebellar deafferentation temporally accelerated PC death from the natural 6-8 week time-course to 1-2 weeks in the shaker mutant rat. The present study posits that IO chemoablation leads to the accelerated and augmented upregulation of the executioner active caspase-3 that triggers apoptosis of at-risk PCs throughout the ordinary phenotypic manifestation of the shaker mutation., Methods: Immunofluorescence and double labeling for calbindin and active caspase-3 were used in vermal cerebellar sections from IO-chemoablated rats to demonstrate the effect of IO chemoablation on active caspase-3 expression in at-risk PCs., Results: Active caspase-3 expression was enhanced in the anterior degeneration (ADC) and posterior degeneration (PDC) compartments to reach a peak in both degeneration compartments at 24 h following the injections for IO chemoablation., Discussion: Consequently, it can be deduced that active caspase-3 expression in shaker mutant rats is modifiable suggesting the possibility of targeting it therapeutically in an attempt to rescue PCs from death. Abbreviation PC: Purkinje cell; IO: inferior olive; ADC: Anterior degeneration compartment; PDC: Posterior degeneration compartment; ISC: Intermediate survival compartment; FNSC: Flocculonodular survival compartment.

A set of glutamylases and deglutamylases controls levels of tubulin polyglutamylation, a prominent post-translational modification of neuronal microtubules. Defective tubulin polyglutamylation was first linked to neurodegeneration in the Purkinje cell degeneration ( pcd ) mouse, which lacks deglutamylase CCP1, displays massive cerebellar atrophy, and accumulates abnormally glutamylated tubulin in degenerating neurons. We found biallelic rare and damaging variants in the gene encoding CCP1 in 13 individuals with infantile-onset neurodegeneration and confirmed the absence of functional CCP1 along with dysregulated tubulin polyglutamylation. The human disease mainly affected the cerebellum, spinal motor neurons, and peripheral nerves. We also demonstrate previously unrecognized peripheral nerve and spinal motor neuron degeneration in pcd mice, which thus recapitulated key features of the human disease. Our findings link human neurodegeneration to tubulin polyglutamylation, entailing this post-translational modification as a potential target for drug development for neurodegenerative disorders., (© 2018 The Authors.)

Inositol hexakisphosphate kinases (IP6Ks) regulate various biological processes. Among pyrophosphates generated by IP6Ks, diphosphoinositol pentakisphosphate (IP7), and bis-diphosphoinositol tetrakisphosphate have been extensively characterized. IP7 is produced in mammals by a family of inositol hexakisphosphate kinases, IP6K1, IP6K2, and IP6K3, which have distinct biological functions. We report that IP6K2 binds protein 4.1.N with high affinity and specificity. Nuclear translocation of 4.1N, which is required for its principal functions, is dependent on IP6K2. Both of these proteins are highly expressed in granule cells of the cerebellum where their interaction regulates Purkinje cell morphology and cerebellar synapses. The deletion of IP6K2 in male/female mice elicits substantial defects in synaptic influences of granule cells upon Purkinje cells as well as notable impairment of locomotor function. Moreover, the disruption of IP6K2-4.1N interactions impairs cell viability. Thus, IP6K2 and its interaction with 4.1N appear to be major determinants of cerebellar disposition and psychomotor behavior. SIGNIFICANCE STATEMENT Inositol phosphates are produced by a family of inositol hexakisphosphate kinases (IP6Ks)-IP6K1, IP6K2, and IP6K3. Of these, the physiological roles of IP6K2 in the brain have been least characterized. In the present study, we report that IP6K2 binds selectively to the neuronal protein 4.1N. Both of these proteins are highly expressed in granule cells of the cerebellum. Using IP6K2 knock-out (KO) mice, we establish that IP6K2-4.1N interactions in granule cells regulate Purkinje cell morphology, the viability of cerebellar neurons, and psychomotor behavior., (Copyright © 2018 the authors 0270-6474/18/387409-11$15.00/0.)

Editing domains of aminoacyl tRNA synthetases correct tRNA charging errors to maintain translational fidelity. A mutation in the editing domain of alanyl tRNA synthetase (AlaRS) in Aars sti mutant mice results in an increase in the production of serine-mischarged tRNA Ala and the degeneration of cerebellar Purkinje cells. Here, using positional cloning, we identified Ankrd16, a gene that acts epistatically with the Aars sti mutation to attenuate neurodegeneration. ANKRD16, a vertebrate-specific protein that contains ankyrin repeats, binds directly to the catalytic domain of AlaRS. Serine that is misactivated by AlaRS is captured by the lysine side chains of ANKRD16, which prevents the charging of serine adenylates to tRNA Ala and precludes serine misincorporation in nascent peptides. The deletion of Ankrd16 in the brains of Aars sti/sti mice causes widespread protein aggregation and neuron loss. These results identify an amino-acid-accepting co-regulator of tRNA synthetase editing as a new layer of the machinery that is essential to the prevention of severe pathologies that arise from defects in editing.

Increasing evidence suggests that synapse dysfunctions are a major determinant of several neurodevelopmental and neurodegenerative diseases. Here we identify protein kinase N1 (PKN1) as a novel key player in fine-tuning the balance between axonal outgrowth and presynaptic differentiation in the parallel fiber-forming (PF-forming) cerebellar granule cells (Cgcs). Postnatal Pkn1-/- animals showed a defective PF-Purkinje cell (PF-PC) synapse formation. In vitro, Pkn1-/- Cgcs exhibited deregulated axonal outgrowth, elevated AKT phosphorylation, and higher levels of neuronal differentiation-2 (NeuroD2), a transcription factor preventing presynaptic maturation. Concomitantly, Pkn1-/- Cgcs had a reduced density of presynaptic sites. By inhibiting AKT with MK-2206 and siRNA-mediated knockdown, we found that AKT hyperactivation is responsible for the elongated axons, higher NeuroD2 levels, and reduced density of presynaptic specifications in Pkn1-/- Cgcs. In line with our in vitro data, Pkn1-/- mice showed AKT hyperactivation, elevated NeuroD2 levels, and reduced expression of PF-PC synaptic markers during stages of PF maturation in vivo. The long-term effect of Pkn1 knockout was further seen in cerebellar atrophy and mild ataxia. In summary, our results demonstrate that PKN1 functions as a developmentally active gatekeeper of AKT activity, thereby fine-tuning axonal outgrowth and presynaptic differentiation of Cgcs and subsequently the correct PF-PC synapse formation.

Among the many types of neurons expressing protein kinase C (PKC) enzymes, cerebellar Purkinje neurons are particularly reliant on appropriate PKC activity for maintaining homeostasis. The importance of PKC enzymes in Purkinje neuron health is apparent as mutations in PRKCG (encoding PKCγ) cause cerebellar ataxia. PRKCG has also been identified as an important node in ataxia gene networks more broadly, but the functional role of PKC in other forms of ataxia remains unexplored, and the mechanisms by which PKC isozymes regulate Purkinje neuron health are not well understood. Here, we investigated how PKC activity influences neurodegeneration in inherited ataxia. Using mouse models of spinocerebellar ataxia type 1 (SCA1) and 2 (SCA2) we identify an increase in PKC-mediated substrate phosphorylation in two different forms of inherited cerebellar ataxia. Normalizing PKC substrate phosphorylation in SCA1 and SCA2 mice accelerates degeneration, suggesting that the increased activity observed in these models is neuroprotective. We also find that increased phosphorylation of PKC targets limits Purkinje neuron membrane excitability, suggesting that PKC activity may support Purkinje neuron health by moderating excitability. These data suggest a functional role for PKC enzymes in ataxia gene networks, and demonstrate that increased PKC activity is a protective modifier of degeneration in inherited cerebellar ataxia.

The function of mitochondria depends on ubiquitously expressed and evolutionary conserved m-AAA proteases in the inner membrane. These ATP-dependent peptidases form hexameric complexes built up of homologous subunits. AFG3L2 subunits assemble either into homo-oligomeric isoenzymes or with SPG7 (paraplegin) subunits into hetero-oligomeric proteolytic complexes. Mutations in AFG3L2 are associated with dominant spinocerebellar ataxia (SCA28) characterized by the loss of Purkinje cells, whereas mutations in SPG7 cause a recessive form of hereditary spastic paraplegia (HSP7) with motor neurons of the cortico-spinal tract being predominantly affected. Pleiotropic functions have been assigned to m-AAA proteases, which act as quality control and regulatory enzymes in mitochondria. Loss of m-AAA proteases affects mitochondrial protein synthesis and respiration and leads to mitochondrial fragmentation and deficiencies in the axonal transport of mitochondria. Moreover m-AAA proteases regulate the assembly of the mitochondrial calcium uniporter (MCU) complex. Impaired degradation of the MCU subunit EMRE in AFG3L2-deficient mitochondria results in the formation of deregulated MCU complexes, increased mitochondrial calcium uptake and increased vulnerability of neurons for calcium-induced cell death. A reduction of calcium influx into the cytosol of Purkinje cells rescues ataxia in an AFG3L2-deficient mouse model. In this review, we discuss the relationship between the m-AAA protease and mitochondrial calcium homeostasis and its relevance for neurodegeneration and describe a novel mouse model lacking MCU specifically in Purkinje cells. Our results pledge for a novel view on m-AAA proteases that integrates their pleiotropic functions in mitochondria to explain the pathogenesis of associated neurodegenerative disorders.

Ethanol and age-induced pathologies of the Purkinje neuron (PN) may result from histone deacetylases (HDACs), enzymes which repress transcription through coiling of the DNA. The purposes of this study were to investigate expression patterns of Class 1 and IIa HDACs in PN and the effects of aging and alcohol on the density of HDACs and histone acetylation in PN. Ninety, eight month old rats (30/diet) were fed a liquid ethanol, liquid control, or rat chow diet for 10, 20, or 40weeks (30/treatment duration). Double immunocytochemical labeling on tissue sections from these rats used antibodies against HDAC isoforms or acetylated histones, and calbindin, a marker for PN. Fluorescent intensities were also measured. Results showed a significant age but not an alcohol-related decrease in the densities of HDACs 2, 3, and 7. In contrast, there were age related-increases in the densities of phosphorylated form of HDAC (4, 5, 7) PN and in PN nuclei expressing HDAC 7. There were also a trend towards ethanol-induced inhibition of acetylation as the density of AH2b PN nuclei and AH3 and AH2b fluorescent intensity was significantly lower in the EF compared to the PF rats. This study presents unique data concerning which HDACs are commonly expressed in PN and indicates that aging rather than lengthy alcohol expression alters expression of the HDACs studied here. These results also suggest that lengthy ethanol consumption may inhibit histone deacetylation in PN., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Cyclin-dependent kinase 5 (Cdk5) is recognized as a unique member among other Cdks due to its versatile roles in many biochemical processes in the nervous system. The proper development of neuronal dendrites is required for the formation of complex neural networks providing the physiological basis of various neuronal functions. We previously reported that sparse dendrites were observed on cultured Cdk5-null Purkinje cells and Purkinje cells in Wnt1 cre -mediated Cdk5 conditional knockout (KO) mice. In the present study, we generated L7 cre -mediated p35; p39 double KO (L7 cre -p35 f/f ; p39 -/- ) mice whose Cdk5 activity was eliminated specifically in Purkinje cells of the developing cerebellum. Consequently, these mice exhibited defective Purkinje cell migration, motor coordination deficiency and a Purkinje dendritic abnormality similar to what we have observed before, suggesting that dendritic growth of Purkinje cells was cell-autonomous in vivo. We found that mixed and overlay cultures of WT cerebellar cells rescued the dendritic deficits in Cdk5-null Purkinje cells, however, indicating that Purkinje cell dendritic development was also supported by non-cell-autonomous factors. We then again rescued these abnormalities in vitro by applying exogenous brain-derived neurotrophic factor (BDNF). Based on the results from culture experiments, we attempted to rescue the developmental defects of Purkinje cells in L7 cre -p35 f/f ; p39 -/- mice by using a TrkB agonist. We observed partial rescue of morphological defects of dendritic structures of Purkinje cells. These results suggest that Cdk5 activity is required for Purkinje cell dendritic growth in cell-autonomous and non-cell-autonomous manners. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1175-1187, 2017., (© 2017 Wiley Periodicals, Inc.)

Following activation of Gq protein-coupled receptors, phospholipase C yields a pair of second messengers: diacylglycerol (DG) and inositol 1,4,5-trisphosphate. Diacylglycerol kinase (DGK) phosphorylates DG to produce phosphatidic acid, another second messenger. Of the DGK family, DGKε is the only DGK isoform that exhibits substrate specificity for DG with an arachidonoyl acyl chain at the sn-2 position. Recently, we demonstrated that hydrophobic residues in the N-terminus of DGKε play an important role in targeting the endoplasmic reticulum in transfected cells. However, its cellular expression and subcellular localization in the brain remain elusive. In the present study, we investigate this issue using specific DGKε antibody. DGKε was richly expressed in principal neurons of higher brain regions, including pyramidal cells in the hippocampus and neocortex, medium spiny neurons in the striatum and Purkinje cells in the cerebellum. In Purkinje cells, DGKε was localized to the subsurface cisterns and colocalized with inositol 1,4,5-trisphosphate receptor-1 in dendrites and axons. In dendrites of Purkinje cells, DGKε was also distributed in close apposition to DG lipase-α, which catalyzes arachidonoyl-DG to produce 2-arachidonoyl glycerol, a major endocannabinoid in the brain. Behaviorally, DGKε-knockout mice exhibited hyper-locomotive activities and impaired motor coordination and learning. These findings suggest that DGKε plays an important role in neuronal and brain functions through its distinct neuronal expression and subcellular localization and also through coordinated arrangement with other molecules involving the phosphoinositide signaling pathway.

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.

Telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase, an enzyme that elongates telomeres at the ends of chromosomes during DNA replication. Recently, it was shown that TERT has additional roles in cell survival, mitochondrial function, DNA repair, and Wnt signaling, all of which are unrelated to telomeres. Here, we demonstrate that TERT is enriched in Purkinje neurons, but not in the granule cells of the adult mouse cerebellum. TERT immunoreactivity in Purkinje neurons is present in the nucleus, mitochondria, and cytoplasm. Furthermore, TERT co-localizes with mitochondrial markers, and immunoblot analysis of protein extracts from isolated mitochondria and synaptosomes confirmed TERT localization in mitochondria. TERT expression in Purkinje neurons increased significantly in response to two stressors: a sub-lethal dose of X-ray radiation and exposure to a high glutamate concentration. While X-ray radiation increased TERT levels in the nucleus, glutamate exposure elevated TERT levels in mitochondria. Our findings suggest that in mature Purkinje neurons, TERT is present both in the nucleus and in mitochondria, where it may participate in adaptive responses of the neurons to excitotoxic and radiation stress.

Aims: Cerebellar ataxia is common in patients with mitochondrial disease, and despite previous neuropathological investigations demonstrating vulnerability of the olivocerebellar pathway in patients with mitochondrial disease, the exact neurodegenerative mechanisms are still not clear. We use quantitative quadruple immunofluorescence to enable precise quantification of mitochondrial respiratory chain protein expression in Purkinje cell bodies and their synaptic terminals in the dentate nucleus., Methods: We investigated NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 13 protein expression in 12 clinically and genetically defined patients with mitochondrial disease and ataxia and 10 age-matched controls. Molecular genetic analysis was performed to determine heteroplasmy levels of mutated mitochondrial DNA in Purkinje cell bodies and inhibitory synapses., Results: Our data reveal that complex I deficiency is present in both Purkinje cell bodies and their inhibitory synapses which surround dentate nucleus neurons. Inhibitory synapses are fewer and enlarged in patients which could represent a compensatory mechanism. Mitochondrial DNA heteroplasmy demonstrated similarly high levels of mutated mitochondrial DNA in cell bodies and synapses., Conclusions: This is the first study to use a validated quantitative immunofluorescence technique to determine complex I expression in neurons and presynaptic terminals, evaluating the distribution of respiratory chain deficiencies and assessing the degree of morphological abnormalities affecting synapses. Respiratory chain deficiencies detected in Purkinje cell bodies and their synapses and structural synaptic changes are likely to contribute to altered cerebellar circuitry and progression of ataxia., (© 2015 The Authors. Neuropathology and Applied Neurobiology published by John Wiley & Sons Ltd on behalf of British Neuropathological Society.)

Background: The cerebellum is responsible for coordinating motor functions and has a unique laminated architecture. Purkinje cells are inhibitory neurons and represent the only output from the cerebellar cortex. Tyrosine hydroxylase (TH) is the key enzyme for the synthesis of catecholamines, including dopamine and noradrenaline, and it is normally not expressed in cerebellar neurons., Results: We report here that the low-density lipoprotein receptors (Lrp) 5 and 6, Wnt co-receptors, are required for the development of the cerebellum and for suppressing ectopic TH expression in Purkinje cells. Simultaneous inactivation of Lrp 5 and 6 by Nestin-Cre results in defective lamination and foliation of the cerebellum during postnatal development. Surprisingly, TH is ectopically expressed by Purkinje cells, although they still keep its other neurochemical characteristics. These phenotypes are also observed in the cerebellum of GFAP-Cre;β-catenin(flox/flox) mice, and AAV2-Cre-mediated gene deletion leads to ectopic TH expression in Purkinje cells of β-catenin(flox/flox) mice as well., Conclusions: Our results revealed a new role of the canonical Lrp5/6-β-catenin pathway in regulating the morphogenesis of the cerebellum during postnatal development.

The avian cerebellum is organized into multiple longitudinal stripes defined by expression profiles of aldolase C (zebrin II) in Purkinje cells. The relationship between the aldolase C striped pattern and the olivocerebellar projection pattern is crucial in understanding cerebellar functional compartmentalization. We identified all aldolase C stripes across all lobules with the serial section alignment analysis method and then looked at this relationship by anterograde and retrograde labeling of olivocerebellar axons in the chick cerebellum. Aldolase C stripes were generally consistent and continuous from lobule I through VII and to the medial part of lobules VIII-IXb. The dorsal and ventral lamellas (DL, VL) of the inferior olive projected to the stripes in these areas with a simple mediolateral topographic relation. A few aldolase C stripes appeared at the lateral edge of lobules VI-VIII. Several more stripes were added in the lateral parts of lobules IXa-IXb and IXc-X. The medial column (MC) of the inferior olive projected to the stripes in lobules VIII-X, including the added lateral stripes, with a complex topographic relation. Sharp boundaries between aldolase C-positive and -negative stripes often accompanied a gap in the Purkinje cell layer and bordered topographically distinct groups of axons. Although the compartmental organization of the chick cerebellum is comparable to that of the mammalian cerebellum, several significant differences in the organization suggest partly separate evolutionary lineages of the mammalian and avian cerebella. We propose that rostral lobules may be evolved by rostral extension of medial stripes from caudal lobules in the avian cerebellum., (© 2015 Wiley Periodicals, Inc.)

The enzyme glucocerebrosidase (GBA) hydrolyses glucosylceramide (GlcCer) in lysosomes. Markedly reduced GBA activity is associated with severe manifestations of Gaucher disease including neurological involvement. Mutations in the GBA gene have recently also been identified as major genetic risk factor for Parkinsonism. Disturbed metabolism of GlcCer may therefore play a role in neuropathology. Besides lysosomal GBA, cells also contain a non-lysosomal glucosylceramidase (GBA2). Given that the two β-glucosidases share substrates, we speculated that over-activity of GBA2 during severe GBA impairment might influence neuropathology. This hypothesis was studied in Niemann-Pick type C (Npc1-/-) mice showing secondary deficiency in GBA in various tissues. Here we report that GBA2 activity is indeed increased in the brain of Npc1-/- mice. We found that GBA2 is particularly abundant in Purkinje cells (PCs), one of the most affected neuronal populations in NPC disease. Inhibiting GBA2 in Npc1-/- mice with a brain-permeable low nanomolar inhibitor significantly improved motor coordination and extended lifespan in the absence of correction in cholesterol and ganglioside abnormalities. This trend was recapitulated, although not to full extent, by introducing a genetic loss of GBA2 in Npc1-/- mice. Our findings point to GBA2 activity as therapeutic target in NPC.

The atypical cyclin-dependent kinase 5 (Cdk5) serves an array of different functions in cell biology. Among these are axonal guidance, regulation of intercellular contacts, cell differentiation, and prosurvival signaling. The variance of these functions suggests that Cdk5 activation comes to pass in different cellular compartments. The kinase activity, half-life, and substrate specificity of Cdk5 largely depend on specific activators, such as p25, p35, p39, and cyclin I. We hypothesized that the subcellular distribution of Cdk5 activators also determines the localization of the Cdk5 protein and sets the stage for targeted kinase activity within distinct cellular compartments to suit the varying roles of Cdk5. Cdk5 localization was analyzed in murine kidney and brain slices of wild-type and cyclin I- and/or p35-null mice by immunohistochemistry and in cultured mouse podocytes using immunofluorescence labeling, as well as cell fractionation experiments. The predominance of cyclin I mediates the nuclear localization of Cdk5, whereas the predominance of p35 results in a membranous localization of Cdk5. These findings were further substantiated by overexpression of cyclin I and p35 with altered targeting characteristics in human embryonic kidney 293T cells. These studies reveal that the subcellular localization of Cdk5 is determined by its specific activators. This results in the directed Cdk5 kinase activity in specific cellular compartments dependent on the activator present and allows Cdk5 to serve multiple independent roles., (Copyright © 2015 the American Physiological Society.)

Matrix metalloproteinase-2 (MMP-2) is a highly studied proteolytic enzyme, involved in many detrimental and beneficial functions throughout the body, and also active in the central nervous system (CNS). MMP-2 is profoundly expressed in the developing cerebellum and was recently reported to modulate granule cell proliferation by affecting cell cycle kinetics in cerebella of postnatal day 3 mouse pups. In this report, a two-dimensional difference gel electrophoresis proteomics study was implemented at this postnatal stage and revealed 16 differentially expressed proteins between MMP-2-deficient (MMP-2(-/-)) and wild-type cerebella. Among those, collapsin response mediator protein 1 (CRMP1) could be identified as the most significant differential protein between the two genotypes. Western blot experiments confirmed this finding and further disclosed a significant increase in phosphorylated CRMP1 expression in MMP-2(-/-) cerebella. Strikingly, subsequent immunohistochemical and microscopic analyses revealed an aberrant Purkinje cell (PC) dendritogenesis, possibly related to upregulated (phospho-) CRMP1 levels in these neonatal MMP-2(-/-) animals. Further, detailed morphometric analyses showed persistent PC morphological changes in MMP-2(-/-) mice, from the neonatal stage until adulthood. These were characterized by a reduced growth of PC somata, reduced dendritic tree sizes, and a decreased dendritic arborization. During development, the observed defects were accompanied by a temporarily disturbed parallel fiber and climbing fiber synaptic input on the PCs, while in adult MMP-2(-/-) animals, an increased PC spine density and reduced spine lengths were noted. The observed PC abnormalities might contribute to the mild defects in motor performance, i.e. balance and coordination, detected in adult MMP-2(-/-) mice. Overall, these findings indicate the importance of MMP-2 in CNS development and dendritogenesis, and highlight the importance of a correct developmental wiring for adult brain morphology and function.

Spinocerebellar ataxias (SCAs) are hereditary diseases leading to Purkinje cell degeneration and cerebellar dysfunction. Most forms of SCA are caused by expansion of CAG repeats similar to other polyglutamine disorders such as Huntington's disease. In contrast, in the autosomal dominant SCA-14 the disease is caused by mutations in the protein kinase C gamma (PKCγ) gene which is a well characterized signaling molecule in cerebellar Purkinje cells. The study of SCA-14, therefore, offers the unique opportunity to reveal the molecular and pathological mechanism eventually leading to Purkinje cell dysfunction and degeneration. We have created a mouse model of SCA-14 in which PKCγ protein with a mutation found in SCA-14 is specifically expressed in cerebellar Purkinje cells. We find that in mice expressing the mutated PKCγ protein the morphology of Purkinje cells in cerebellar slice cultures is drastically altered and mimics closely the morphology seen after pharmacological PKC activation. Similar morphological abnormalities were seen in localized areas of the cerebellum of juvenile transgenic mice in vivo. In adult transgenic mice there is evidence for some localized loss of Purkinje cells but there is no overall cerebellar atrophy. Transgenic mice show a mild cerebellar ataxia revealed by testing on the rotarod and on the walking beam. Our findings provide evidence for both an increased PKCγ activity in Purkinje cells in vivo and for pathological changes typical for cerebellar disease thus linking the increased and dysregulated activity of PKCγ tightly to the development of cerebellar disease in SCA-14 and possibly also in other forms of SCA., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Mucolipidosis II (MLII) is a lysosomal storage disorder caused by loss of N-acetylglucosamine-1-phosphotransferase, which tags lysosomal enzymes with a mannose 6-phosphate marker for transport to the lysosome. In MLII, the loss of this marker leads to deficiency of multiple enzymes and non-enzymatic proteins in the lysosome, leading to the storage of multiple substrates. Here we present a novel mouse model of MLII homozygous for a patient mutation in the GNPTAB gene. Whereas the current gene knock-out mouse model of MLII lacks some of the characteristic features of the human disease, our novel mouse model more fully recapitulates the human pathology, showing growth retardation, skeletal and facial abnormalities, increased circulating lysosomal enzymatic activities, intracellular lysosomal storage, and reduced life span. Importantly, MLII behavioral deficits are characterized for the first time, including impaired motor function and psychomotor retardation. Histological analysis of the brain revealed progressive neurodegeneration in the cerebellum with severe Purkinje cell loss as the underlying cause of the ataxic gait. In addition, based on the loss of Npc2 (Niemann-Pick type C 2) protein expression in the brain, the mice were treated with 2-hydroxypropyl-β-cyclodextrin, a drug previously reported to rescue Purkinje cell death in a mouse model of Niemann-Pick type C disease. No improvement in brain pathology was observed. This indicates that cerebellar degeneration is not primarily triggered by loss of Npc2 function. This study emphasizes the value of modeling MLII patient mutations to generate clinically relevant mouse mutants to elucidate the pathogenic molecular pathways of MLII and address their amenability to therapy., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

The enzyme 5α-reductase catalyzes the transformation of progesterone, testosterone, and deoxycorticosterone into 5α-reduced metabolites, which are recognized as neurosteroids in the brain with variable potential neuroactivity. Two isoforms of 5α-reductase were identified in rodents, and, of these, 5α-reductase type 1 (5α-R1) is abundantly expressed in the brain. To understand the multiple influences of neurosteroids in the central nervous system, we need to know their region-specific synthesis. The present study reports the detailed localization of 5α-R1 in the adult rat cerebellum. The occurrence of 5α-R1 was detected by reverse transcription-polymerase chain reaction. The enzyme activity was also detected by thin layer chromatography. Immunocytochemistry showed 5α-R1 immunoreactive cells in all cerebellar layers. Multiple immunolabeling revealed that 5α-R1 was mainly localized in glia, such as astrocytes and oligodendrocytes. The most intense immunoreactivity for 5α-R1 was found in Bergmann glia, and the processes of these glia were associated with dendrites of both Purkinje cells and interneurons in the molecular layer. The 5α-R1 in the cerebellum was expressed consistently throughout different ages and sexes, in both gonadectomized and hypophysectomized rats. Thus, 5α-R1 may contribute to the formation and maintenance of the cerebellar neurons through 5α-reduced metabolites, which are synthesized through a complex interaction between neurons and glia., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Diabetic neuropathy develops on a background of hyperglycemia and an entangled metabolic imbalance. There is increasing evidence of central nervous system involvement in diabetic neuropathy and no satisfactory treatment except maintenance of good glycemic control, thereby highlighting the importance of identifying novel therapeutic targets. Purkinje cells are a class of metabolically specialized active neurons, and degeneration of Purkinje cells is a common feature of inherited ataxias in humans and mice. However, whether Purkinje cells are implicated in diabetic neuropathy development under metabolic stress remains poorly defined. Here, we revealed a novel leucine-rich repeat kinase 2 (LRRK2)-mediated pathway in Purkinje cells that is involved in the pathogenesis of diabetic neuropathy from a 24-week long study of streptozotocin (STZ)-diabetic rats. We found that hyperglycemia, cerebellum proinflammatory cytokines, and chemokines increased markedly in 24-week STZ-diabetic rats. Furthermore, we demonstrated that degeneration of Purkinje cells is characterized by progressive swellings of axon terminals, no autophagosome formation, the reduction of LC3II/LC3I and Lamp2, and accumulation of p62 puncta in 24-week STZ-diabetic rats. Importantly, a higher expression level of LRRK2-mediated hyperphosphorylation of tau along with increased mitochondrial dynamin-like protein (mito-DLP1) was demonstrated in 24-week STZ-diabetic rats. This effect of LRRK2 overexpression induced mitochondrial fragmentation, and reduced mitochondrial protein degradation rates were confirmed in vitro. As a consequence, 24-week STZ-diabetic rats showed mitochondrial dysfunction in cerebellar Purkinje neurons and coordinated motor deficits evaluated by rotarod test. Our findings are to our knowledge the first to suggest that the LRRK2-mediated pathway induces mitochondrial dysfunction and loss of cerebellar Purkinje neurons and, subsequently, may be associated with motor coordination deficits in STZ-diabetic rats. These data may indicate a novel cellular therapeutic target for diabetic neuropathy.

Methylmercury (MeHg), an environmental neurotoxicant, induces site-specific toxicity in the brain. Although oxidative stress has been demonstrated with MeHg toxicity, the site-specific toxicity is not completely understood. Among the cerebellar neurons, cerebellar granule cells (CGCs) appear vulnerable to MeHg, whereas Purkinje cells and molecular layer neurons are resistant. Here, we use a MeHg-intoxicated rat model to investigate these cerebellar neurons for the different causes of susceptibility to MeHg. Rats were exposed to 20 ppm MeHg for 4 weeks and subsequently exhibited neuropathological changes in the cerebellum that were similar to those observed in humans. We first isolated the three cerebellar neuron types using a microdissection system and then performed real-time PCR analyses for antioxidative enzymes. We observed that expression of manganese-superoxide dismutase (Mn-SOD), glutathione peroxidase 1 (GPx1), and thioredoxin reductase 1 (TRxR1) was significantly higher in Purkinje cells and molecular layer neurons than in CGCs. Finally, we performed immunohistochemical analyses on the cerebellum. Immunohistochemistry showed increased expression of Mn-SOD, GPx1, and TRxR1 in Purkinje cells and molecular layer neurons, which was coincident with the mRNA expression patterns. Considering Mn-SOD, GPx1, and TRxR1 are critical for protecting cells against MeHg intoxication, the results indicate that low expression of these antioxidative enzymes increases CGCs vulnerability to MeHg toxicity.

Purpose of the Review: To review the recent advances in the understanding and management of the immune and nonimmune effects of inherited adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) deficiencies., Recent Findings: Abnormal thymocyte development and peripheral T-cell activation in ADA-deficient and PNP-deficient patients cause increased susceptibility to infections and immune dysregulation. The impaired purine homeostasis also damages many other cell types and tissues. Animal studies suggest that defects in surfactant metabolism by alveolar macrophages cause the pulmonary alveolar proteinosis commonly seen in ADA-deficient infants, while toxicity of purine metabolites to cerebellar Purkinje cells may lead to the ataxia frequently observed in PNP deficiency. Patients' outcome with current treatments including enzyme replacement and stem cell transplantations are inferior to those achieved in most severe immunodeficiency conditions. New strategies, including intracellular enzyme replacement, gene therapy and innovative protocols for stem cell transplantations hold great promise for improved outcomes in ADA and PNP deficiency. Moreover, newborn screening and early diagnosis will allow prompt application of these novel treatment strategies, further improving survival and reducing morbidity., Summary: Better understanding of the complex immune and nonimmune effects of ADA and PNP deficiency holds great promise for improved patients' outcome.

Purkinje neurons are a sensitive and specialised cell type important for fine motor movement and coordination. Purkinje cell damage manifests as motor incoordination and ataxia - a prominent feature of many human disorders including spinocerebellar ataxia and Huntington's disease. A correlation between Purkinje degeneration and excess cerebellar levels of tissue-type plasminogen activator (tPA) has been observed in multiple genetically-distinct models of ataxia. Here we show that Purkinje loss in a mouse model of Huntington's disease also correlates with a 200% increase in cerebellar tPA activity. That elevated tPA levels arise in a variety of ataxia models suggests that tPA is a common mediator of Purkinje damage. To address the specific contribution of tPA to cerebellar dysfunction we studied the T4 mice line that overexpresses murine tPA in postnatal neurons through the Thy1.2 gene promoter, which directs preferential expression to Purkinje cells within the cerebellum. Here we show that T4 mice develop signs of cerebellar damage within 10 weeks of birth including atrophy of Purkinje cell soma and dendrites, astrogliosis, reduced molecular layer volume and altered gait. In contrast, T4 mice displayed no evidence of microgliosis, nor any changes in interneuron density, nor alteration in the cerebellar granular neuron layer. Thus, excess tPA levels may be sufficient to cause targeted Purkinje cell degeneration and ataxia. We propose that elevated cerebellar tPA levels exert a common pathway of Purkinje cell damage. Therapeutically lowering cerebellar tPA levels may represent a novel means of preserving Purkinje cell integrity and motor coordination across a wide range of neurodegenerative diseases., (© 2013.)

We previously reported that heat stroke induces autophagy as a protection mechanism against neurodegeneration in the brain. Heme oxygenase (HO)-1 is a stress protein and can be induced by heat stress (HS). Cerebellar Purkinje cells are selectively vulnerable to heat-induced injury. In this study, we first validated an animal model of HS (38°C for 4 h) in which sustained increase of Purkinje cell injury, HO-1 expression up to 24 h post HS (HS₂₄), and hyperthermia reaching a rectal temperature 41.52 ± 0.32 were observed. In subsequent experiments, we investigated the effects of HO-1 on HS-induced Purkinje cell injury. Rats were divided into four groups: one normothermic control group receiving saline vehicle (1 mL/kg, intraperitoneal [i.p.]) and exposed to 25 for 4 h; and three HS groups receiving saline, or HO-1 inducer haemin (30 mg/kg, i.p.) or HO-1 inhibitor tin protoporphyrin (SnPP, 30 mg/kg, i.p.), respectively, at 12 h prior to HS. HS-induced Purkinje cell injury was further enhanced by HO-1 inducer but attenuated by HO-1 inhibitor as evaluated by immunoreactivity of apoptosis marker (active caspase-3) as well as Fluoro-Jade B histochemistry (staining for degenerating neurons), suggesting a detrimental role of HO-1. Interestingly, the protective autophagy was reduced by HO-1 inducer but enhanced by HO-1 inhibitor as demonstrated by autophagy markers including Beclin-1 and microtubule-associated protein light chain 3 in Purkinje cells. Double immunofluorescent labelling of Beclin-1 or 8-hydroxydeoxyguanosine (an oxidative DNA damage marker) with HO-1 immunoreactivity not only demonstrated their co-localization, but also confirmed that HO-1 negatively regulated Beclin-1 but increased oxidative stress in the same Purkinje cell. Taken together, our results indicate that HO-1 aggravates HS injury in cerebellar Purkinje cells. Our findings shed new light on cell damage mechanisms by HS in central nervous system and may help to provide potential therapeutic foci.

The mammalian target of rapamycin (mTOR) assembles into two distinct multi-protein complexes called mTORC1 and mTORC2. Whereas mTORC1 is known to regulate cell and organismal growth, the role of mTORC2 is less understood. We describe two mouse lines that are devoid of the mTORC2 component rictor in the entire central nervous system or in Purkinje cells. In both lines neurons were smaller and their morphology and function were strongly affected. The phenotypes were accompanied by loss of activation of Akt, PKC, and SGK1 without effects on mTORC1 activity. The striking decrease in the activation and expression of several PKC isoforms, the subsequent loss of activation of GAP-43 and MARCKS, and the established role of PKCs in spinocerebellar ataxia and in shaping the actin cytoskeleton strongly suggest that the morphological deficits observed in rictor-deficient neurons are mediated by PKCs. Together our experiments show that mTORC2 has a particularly important role in the brain and that it affects size, morphology, and function of neurons.

Congenital ataxia and mental retardation are mainly caused by variations in the genes that affect brain development. Recent reports have shown that mutations in the CA8 gene are associated with mental retardation and ataxia in humans and ataxia in mice. The gene product, carbonic anhydrase-related protein VIII (CARP VIII), is predominantly present in cerebellar Purkinje cells, where it interacts with the inositol 1,4,5-trisphosphate receptor type 1, a calcium channel. In this study, we investigated the effects of the loss of function of CARP VIII during embryonic development in zebrafish using antisense morpholino oligonucleotides against the CA8 gene. Knockdown of CA8 in zebrafish larvae resulted in a curved body axis, pericardial edema and abnormal movement patterns. Histologic examination revealed gross morphologic defects in the cerebellar region and in the muscle. Electron microscopy studies showed increased neuronal cell death in developing larvae injected with CA8 antisense morpholinos. These data suggest a pivotal role for CARP VIII during embryonic development. Furthermore, suppression of CA8 expression leads to defects in motor and coordination functions, mimicking the ataxic human phenotype. This work reveals an evolutionarily conserved function of CARP VIII in brain development and introduces a novel zebrafish model in which to investigate the mechanisms of CARP VIII-related ataxia and mental retardation in humans.

DOC-2/DAB-2 interacting protein (Dab2IP) is a GTPase activating protein that binds to Disabled-1, a cytosolic adapter protein involved in Reelin signaling and brain development. Dab2IP regulates PI3K-AKT signaling and is associated with metastatic prostate cancer, abdominal aortic aneurysms and coronary heart disease. To date, the physiological function of Dab2IP in the nervous system, where it is highly expressed, is relatively unknown. In this study, we generated a mouse model with a targeted disruption of Dab2IP using a retrovirus gene trap strategy. Unlike reeler mice, Dab2IP knock-down mice did not exhibit severe ataxia or cerebellar hypoplasia. However, Dab2IP deficiency produced a number of cerebellar abnormalities such as a delay in the development of Purkinje cell (PC) dendrites, a decrease in the parallel fiber synaptic marker VGluT1, and an increase in the climbing fiber synaptic marker VGluT2. These findings demonstrate for the first time that Dab2IP plays an important role in dendrite development and regulates the number of synapses in the cerebellum.

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 Ca(2+) channels. We have now studied the role of the plasma membrane Ca(2+)-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.

Inherited defects in purine nucleoside phosphorylase (PNP) cause severe T cell immunodeficiency and progressive neurological dysfunction, yet little is known about the effects of PNP deficiency on the brain. PNP-KO mice display metabolic and immune anomalies similar to those observed in patients. Our objectives were to characterize brain abnormalities in PNP-KO mice and determine whether restoring PNP activity prevents these abnormalities. We analyzed structural brain defects in PNP-KO mice by magnetic resonance imaging, while assessing motor deficits using the accelerating rotarod and stationary balance beam tests. We detected morphological abnormalities and apoptosis in the cerebellum of PNP-KO mice by hematoxylin and eosin, electron microscopy, TUNEL and activated caspase 3 staining. We treated PNP-KO mice with PNP fused to the HIV-TAT protein transduction domain (TAT-PNP) from birth or from 4 weeks of age. Magnetic resonance imaging revealed a smaller than normal cerebellum in PNP-KO mice. PNP-KO mice displayed motor abnormalities including rapid fall from the rotating rod and frequent slips from the balance beam. The cerebellum of PNP-KO mice contained reduced purkinje cells (PC), which were irregular in shape and had degenerated dendrites. PC from the cerebellum of PNP-KO mice, expanded ex vivo, demonstrated increased apoptosis, which could be corrected by supplementing cultures with TAT-PNP. TAT-PNP injections restored PNP activity in the cerebellum of PNP-KO mice. TAT-PNP from birth, but not treatment initiated at 4 weeks of age, prevented the cerebellar PC damage and motor deficits. We conclude that PNP deficiency cause cerebellar abnormalities, including PC damage and progressive motor deficits. TAT-PNP treatment from birth can prevent the neurological abnormalities in PNP-KO mice., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Dendrite development occupies a central position in the formation of nervous system. However, whether lysosomal degradative function is required for dendritogenesis of neurons remains unknown. We have recently demonstrated the critical role of Vps18 in the lysosomal degradation pathway in mice. Here, we report that Vps18 deficiency severely blocks the dendrite development of Pukinje cells but not cerebral cortical neurons. Furthermore, we also demonstrate that the lysyl oxidase (Lox) protein is degraded through lysosome and accumulated in the Vps18 deficient cerebellum but not in cerebral cortices. Our results suggest that lysosome regulates dendritogenesis of Purkinje cells though degrading Lox., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Many cellular processes involve a small number of molecules and undergo stochastic fluctuations in their levels of activity. Cerebellar long-term depression (LTD) is a form of synaptic plasticity expressed as a reduction in the number of synaptic AMPA receptors (AMPARs) in Purkinje cells. We developed a stochastic model of the LTD signaling network, including a PKC-ERK-cPLA(2) positive feedback loop and mechanisms of AMPAR trafficking, and tuned the model to replicate calcium uncaging experiments. The signaling network activity in single synapses switches between two discrete stable states (LTD and non-LTD) in a probabilistic manner. The stochasticity of the signaling network causes threshold dithering and allows at the macroscopic level for many different and stable mean magnitudes of depression. The probability of LTD occurrence in a single spine is only modulated by the concentration and duration of the signal used to trigger it, and inputs with the same magnitude can give rise to two different responses; there is no threshold for the input signal. The stochasticity is intrinsic to the signaling network and not mostly dependent on noise in the calcium input signal, as has been suggested previously. The activities of the ultrasensitive ERK and of cPLA(2) undergo strong stochastic fluctuations. Conversely, PKC, which acts as a noise filter, is more constantly activated. Systematic variation of the biochemical population size demonstrates that threshold dithering and the absence of spontaneous LTD depend critically on the number of molecules in a spine, indicating constraints on spine size in Purkinje cells.

Telomerase promotes tissue regeneration by delaying the entrance of cells into senescence. Studies performed on cells or animals overexpressing telomerase reverse transcriptase (TERT), the catalytic subunit of telomerase, have revealed that TERT exhibits antiapoptotic effects in neurons. However, it is not clear whether endogenous TERT possesses these functions as well. Here we demonstrate the presence of active telomerase in the cytoplasm and nucleus of cerebellar Purkinje neurons of adult and old mice. TERT protein levels are reduced with age, whereas in the nucleus TERT activity is increased. These findings suggest that telomerase plays a role in the aging of nondividing cells.

The Purkinje cell degeneration (pcd) mouse has a disruption in the gene encoding cytosolic carboxypeptidase 1 (CCP1). This study tested two proposed functions of CCP1: degradation of intracellular peptides and processing of tubulin. Overexpression (2-3-fold) or knockdown (80-90%) of CCP1 in human embryonic kidney 293T cells (HEK293T) did not affect the levels of most intracellular peptides but altered the levels of α-tubulin lacking two C-terminal amino acids (delta2-tubulin) ≥ 5-fold, suggesting that tubulin processing is the primary function of CCP1, not peptide degradation. Purified CCP1 produced delta2-tubulin from purified porcine brain α-tubulin or polymerized HEK293T microtubules. In addition, CCP1 removed Glu residues from the polyglutamyl side chains of porcine brain α- and β-tubulin and also generated a form of α-tubulin with two C-terminal Glu residues removed (delta3-tubulin). Consistent with this, pcd mouse brain showed hyperglutamylation of both α- and β-tubulin. The hyperglutamylation of α- and β-tubulin and subsequent death of Purkinje cells in pcd mice was counteracted by the knock-out of the gene encoding tubulin tyrosine ligase-like-1, indicating that this enzyme hyperglutamylates α- and β-tubulin. Taken together, these results demonstrate a role for CCP1 in the processing of Glu residues from β- as well as α-tubulin in vitro and in vivo.

Cerebellin precursor protein 1 (Cbln1) is the prototype of a family of secreted neuronal glycoproteins (Cbln1-4) and its genetic elimination results in synaptic alterations in cerebellum (CB) and striatum. In CB, Cbln1 acts as a bi-functional ligand bridging pre-synaptic β-neurexins on granule cells to post-synaptic Grid2 on Purkinje neurons. Although much is known concerning the action of Cbln1, little is known of the function of its other family members. Here, we show that Cbln1 and Cbln2 have similar binding activities to β-neurexins and Grid2 and the targeted ectopic expression of Cbln2 to Purkinje cells in transgenic mice rescues the cerebellar deficits in Cbln1-null animals: suggesting that the two proteins have redundant function mediated by their common receptor binding properties. Cbln1 and Cbln2 are also co-expressed in the endolysosomal compartment of the thalamic neurons responsible for the synaptic alterations in striatum of Cbln1-null mice. Therefore, to determine whether the two family members have similar functions, we generated Cbln2-null mice. Cbln2-null mice do not show the synaptic alterations evident in striatum of Cbln1-null mice. Thus, Cbln2 can exhibit functional redundancy with Cbln1 in CB but it does not have the same properties as Cbln1 in thalamic neurons, implying one or both utilize different receptors/mechanisms in this brain region., (© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)

Nitric oxide has been investigated widely both during neurodevelopment and in neurological diseases. However, whilst it has been established that nitric oxide-producing enzymes of nitric oxide synthase family are expressed in cerebellar Purkinje neurons, the effects of nitric oxide on the viability and morphology of these neurons remain unknown. Here, we have demonstrated that the activity of neuronal nitric oxide synthase, but not the inducible or endothelial forms of this enzyme, is required to support the survival of a proportion of cerebellar Purkinje neurons in vitro. We discovered that donation of high concentrations of exogenous nitric oxide reduces Purkinje neuron survival in culture and that peroxynitrite is also toxic to these cells. Finally, we demonstrated that exogenous nitric oxide and peroxynitrite reduce both the magnitude and the complexity of the neurite arbour extended by cerebellar Purkinje neurons. Taken together, these findings reveal that whilst a low level of endogenous nitric oxide, released by the activity of neuronal nitric oxide synthase, is beneficial to cerebellar Purkinje neurons in vitro, high levels of exogenous nitric oxide and peroxynitrite are detrimental to both the survival of these neurons and to their ability to extend processes and form functional neural networks.

Astrogliosis, oligodendroglial death and motor deficits have been observed in the offspring of female rats that had their uterine arteries clamped at the 18(th) gestational day. Since nitric oxide has important roles in several inflammatory and developmental events, here we evaluated NADPH-diaphorase (NADPH-d) distribution in the cerebellum of rats submitted to this hypoxia-ischemia (HI) model. At postnatal (P) day 9, Purkinje cells of SHAM and non-manipulated (NM) animals showed NADPH-d+ labeling both in the cell body and dendritic arborization in folia 1 to 8, while HI animals presented a weaker labeling in both cellular structures. NADPH-d+ labeling in the molecular (ML), and in both the external and internal granular layer, was unaffected by HI at this age. At P23, labeling in Purkinje cells was absent in all three groups. Ectopic NADPH-d+ cells in the ML of folia 1 to 4 and folium 10 were present exclusively in HI animals. This labeling pattern was maintained up to P90 in folium 10. In the cerebellar white matter (WM), at P9 and P23, microglial (ED1+) NADPH-d+ cells, were observed in all groups. At P23, only HI animals presented NADPH-d labeling in the cell body and processes of reactive astrocytes (GFAP+). At P9 and P23, the number of NADPH-d+ cells in the WM was higher in HI animals than in SHAM and NM ones. At P45 and at P90 no NADPH-d+ cells were observed in the WM of the three groups. Our results indicate that HI insults lead to long-lasting alterations in nitric oxide synthase expression in the cerebellum. Such alterations in cerebellar differentiation might explain, at least in part, the motor deficits that are commonly observed in this model.

Background: Chaperone-mediated autophagy (CMA) is a selective autophagy-lysosome protein degradation pathway. The role of CMA in normal neuronal functions and in neural disease pathogenesis remains unclear, in part because there is no available method to monitor CMA activity at the single-cell level., Methodology/principal Findings: We sought to establish a single-cell monitoring method by visualizing translocation of CMA substrates from the cytosol to lysosomes using the HaloTag (HT) system. GAPDH, a CMA substrate, was fused to HT (GAPDH-HT); this protein accumulated in the lysosomes of HeLa cells and cultured cerebellar Purkinje cells (PCs) after labeling with fluorescent dye-conjugated HT ligand. Lysosomal accumulation was enhanced by treatments that activate CMA and prevented by siRNA-mediated knockdown of LAMP2A, a lysosomal receptor for CMA, and by treatments that inactivate CMA. These results suggest that lysosomal accumulation of GAPDH-HT reflects CMA activity. Using this method, we revealed that mutant γPKC, which causes spinocerebellar ataxia type 14, decreased CMA activity in cultured PCs., Conclusion/significance: In the present study, we established a novel fluorescent-based method to evaluate CMA activity in a single neuron. This novel method should be useful and valuable for evaluating the role of CMA in various neuronal functions and neural disease pathogenesis.

Cerebellar Purkinje cells (PCs) express a large amount of the γ isoform of protein kinase C (PKCγ) and a modest level of PKCα. The PKCγ is involved in the pruning of climbing fiber (CF) synapses from developing PCs, and PKCα plays a critical role in long-term depression (LTD) at parallel fiber (PF)-PC synapses. Moreover, the PKC signaling in PCs negatively modulates the nonselective transient receptor potential cation channel type 3 (TRPC3), the opening of which elicits slow EPSCs at PF-PC synapses. Autosomal dominant spinocerebellar ataxia type 14 (SCA14) is caused by mutations in PKCγ. To clarify the pathology of this disorder, mutant (S119P) PKCγ tagged with GFP was lentivirally expressed in developing and mature mouse PCs in vivo, and the effects were assessed 3 weeks after the injection. Mutant PKCγ-GFP aggregated in PCs without signs of degeneration. Electrophysiology results showed impaired pruning of CF synapses from developing PCs, failure of LTD expression, and increases in slow EPSC amplitude. We also found that mutant PKCγ colocalized with wild-type PKCγ, which suggests that mutant PKCγ acts in a dominant-negative manner on wild-type PKCγ. In contrast, PKCα did not colocalize with mutant PKCγ. The membrane residence time of PKCα after depolarization-induced translocation, however, was significantly decreased when it was present with the mutant PKCγ construct. These results suggest that mutant PKCγ in PCs of SCA14 patients could differentially impair the membrane translocation kinetics of wild-type γ and α PKCs, which would disrupt synapse pruning, synaptic plasticity, and synaptic transmission.

Several studies have shown that Purkinje cells die by apoptosis in organotypic slice cultures from postnatal 3-day-old (P3) mice. This cell death is age-dependent and has been proposed as indirect evidence for the programmed Purkinje cell death occurring in in vivo cerebellum. Here, we studied whether c-jun N-terminal kinase (JNK) and p38 kinase pathways contribute to the Purkinje cell death observed in cerebellar slice cultures obtained from P3 mice. Slice culture treatment with D-JNKI1 or SB203580, respectively inhibitors of JNK and p38 MAP kinases, results in a better survival of Purkinje cells. Interestingly, the combined treatment with the two inhibitors potentiated single treatment effects. These results suggest that p38 and JNK pathways might be differently implicated in this Purkinje cell death. Time course experiments found p38 activation immediately post-slicing, whereas JNK activation was detected only 2 h after the culture. We hypothesize that p38 activation might be due to the "sliced condition," and JNK activation might be more specific to P3 age-dependent cell death. The study of JNK and p38 activation in cerebellar lysates from P0 slice culture confirmed JNK activation being specific for the P3 explants, whereas p38 is activated both from P0 and P3 cerebellar slice culture lysates. These results suggest that p38 is activated by the slicing, whereas JNK activation is related to developmental Purkinje cell death.

Angelman syndrome (AS) is a severe neurological disorder caused by a deficiency of ubiquitin protein ligase E3A (UBE3A), but the pathophysiology of the disease remains unknown. We now report that in the brains of AS mice in which the maternal UBE3A allele is mutated (m-) and the paternal allele is potentially inactivated by imprinting (p+) (UBE3A m-\p+), the mitochondria are abnormal and exhibit a partial oxidative phosphorylation (OXPHOS) defect. Electron microscopy of the hippocampal region of the UBE3A m-\p+ mice (n=6) reveals small, dense mitochondria with altered cristae, relative to wild-type littermates (n=6) and reduced synaptic vesicle density. The specific activity of OXPHOS complex III is reduced in whole brain mitochondria in UBE3A m-\p+ (n=5) mice versus wild-type littermates (n=5). Therefore, mitochondrial dysfunction may contribute to the pathophysiology of Angelman syndrome., (Copyright © 2009 Elsevier Ireland Ltd. All rights reserved.)