Purkinje Cell Synapses Research Articles

Distinct functional domains of Dystroglycan regulate inhibitory synapse formation and maintenance in cerebellar Purkinje cells.

Dystroglycan is a cell adhesion molecule that localizes to synapses throughout the nervous system. While Dystroglycan is required to maintain inhibitory synapses from cerebellar molecular layer interneurons (MLIs) onto Purkinje cells (PCs) whether initial synaptogenesis during development is dependent on Dystroglycan has not been examined. We show that conditional deletion of Dystroglycan from Purkinje cells prior to synaptogenesis results in impaired MLI:PC synapse formation and function due to reduced presynaptic inputs and abnormal postsynaptic GABAA receptor clustering. Using genetic manipulations that disrupt glycosylation of Dystroglycan or truncate its cytoplasmic domain, we show that Dystroglycan's role in synapse function requires both extracellular and intracellular interactions, whereas synapse formation requires only extracellular interactions. Together, these findings provide molecular insight into the mechanism of inhibitory synapse formation and maintenance in cerebellar cortex.

Mesoscale simulations predict the role of synergistic cerebellar plasticity during classical eyeblink conditioning.

According to the motor learning theory by Albus and Ito, synaptic depression at the parallel fibre to Purkinje cells synapse (pf-PC) is the main substrate responsible for learning sensorimotor contingencies under climbing fibre control. However, recent experimental evidence challenges this relatively monopolistic view of cerebellar learning. Bidirectional plasticity appears crucial for learning, in which different microzones can undergo opposite changes of synaptic strength (e.g. downbound microzones-more likely depression, upbound microzones-more likely potentiation), and multiple forms of plasticity have been identified, distributed over different cerebellar circuit synapses. Here, we have simulated classical eyeblink conditioning (CEBC) using an advanced spiking cerebellar model embedding downbound and upbound modules that are subject to multiple plasticity rules. Simulations indicate that synaptic plasticity regulates the cascade of precise spiking patterns spreading throughout the cerebellar cortex and cerebellar nuclei. CEBC was supported by plasticity at the pf-PC synapses as well as at the synapses of the molecular layer interneurons (MLIs), but only the combined switch-off of both sites of plasticity compromised learning significantly. By differentially engaging climbing fibre information and related forms of synaptic plasticity, both microzones contributed to generate a well-timed conditioned response, but it was the downbound module that played the major role in this process. The outcomes of our simulations closely align with the behavioural and electrophysiological phenotypes of mutant mice suffering from cell-specific mutations that affect processing of their PC and/or MLI synapses. Our data highlight that a synergy of bidirectional plasticity rules distributed across the cerebellum can facilitate finetuning of adaptive associative behaviours at a high spatiotemporal resolution.

Developmental transformation of Ca2+ channel-vesicle nanotopography at a central GABAergic synapse

The coupling between Ca2+ channels and release sensors is a key factor defining the signaling properties of a synapse. However, the coupling nanotopography at many synapses remains unknown, and it is unclear how it changes during development. To address these questions, we examined coupling at the cerebellar inhibitory basket cell (BC)-Purkinje cell (PC) synapse. Biophysical analysis of transmission by paired recording and intracellular pipette perfusion revealed that the effects of exogenous Ca2+ chelators decreased during development, despite constant reliance of release on P/Q-type Ca2+ channels. Structural analysis by freeze-fracture replica labeling (FRL) and transmission electron microscopy (EM) indicated that presynaptic P/Q-type Ca2+ channels formed nanoclusters throughout development, whereas docked vesicles were only clustered at later developmental stages. Modeling suggested a developmental transformation from a more random to a more clustered coupling nanotopography. Thus, presynaptic signaling developmentally approaches a point-to-point configuration, optimizing speed, reliability, and energy efficiency of synaptic transmission.

Ablation of KIF2C in Purkinje cells impairs metabotropic glutamate receptor trafficking and motor coordination in male mice.

Kinesin family member 2C (KIF2C)/mitotic centromere-associated kinesin (MCAK), is thought to be oncogenic as it is involved in tumour progression and metastasis. Moreover, it also plays a part in neurodegenerative conditions like Alzheimer's disease and psychiatric disorders such as suicidal schizophrenia. Our previous study conducted on mice demonstrated that KIF2C is widely distributed in various regions of the brain, and is localized in synaptic spines. Additionally, it regulates microtubule dynamic properties through its own microtubule depolymerization activity, thereby affecting AMPA receptor transport and cognitive behaviour in mice. In this study, we show that KIF2C regulates the transport of mGlu1 receptors in Purkinje cells by binding to Rab8. KIF2C deficiency in Purkinje cells results in abnormal gait, reduced balance ability and motor incoordination in male mice. These data suggest that KIF2C is essential for maintaining normal transport and synaptic function of mGlu1 and motor coordination in mice. KEY POINTS: KIF2C is localized in synaptic spines of hippocampus neurons, and regulates excitatory transmission, synaptic plasticity and cognitive behaviour. KIF2C is extensively expressed in the cerebellum, and we investigated its functions in development and synaptic transmission of cerebellar Purkinje cells. KIF2C deficiency in Purkinje cells alters the expression of metabotropic glutamate receptor 1 (mGlu1) and the AMPA receptor GluA2 subunit at Purkinje cell synapses, and changes excitatory synaptic transmission, but not inhibitory transmission. KIF2C regulates the transport of mGlu1 receptors in Purkinje cells by binding to Rab8. KIF2C deficiency in Purkinje cells affects motor coordination, but not social behaviour in male mice.

Protein Networks Associated with Native Metabotropic Glutamate 1 Receptors (mGlu1) in the Mouse Cerebellum.

The metabotropic glutamate receptor 1 (mGlu1) plays a pivotal role in synaptic transmission and neuronal plasticity. Despite the fact that several interacting proteins involved in the mGlu1 subcellular trafficking and intracellular transduction mechanisms have been identified, the protein network associated with this receptor in specific brain areas remains largely unknown. To identify novel mGlu1-associated protein complexes in the mouse cerebellum, we used an unbiased tissue-specific proteomic approach, namely co-immunoprecipitation followed by liquid chromatography/tandem mass spectrometry analysis. Many well-known protein complexes as well as novel interactors were identified, including G-proteins, Homer, δ2 glutamate receptor, 14-3-3 proteins, and Na/K-ATPases. A novel putative interactor, KCTD12, was further investigated. Reverse co-immunoprecipitation with anti-KCTD12 antibodies revealed mGlu1 in wild-type but not in KCTD12-knock-out homogenates. Freeze-fracture replica immunogold labeling co-localization experiments showed that KCTD12 and mGlu1 are present in the same nanodomain in Purkinje cell spines, although at a distance that suggests that this interaction is mediated through interposed proteins. Consistently, mGlu1 could not be co-immunoprecipitated with KCTD12 from a recombinant mammalian cell line co-expressing the two proteins. The possibility that this interaction was mediated via GABAB receptors was excluded by showing that mGlu1 and KCTD12 still co-immunoprecipitated from GABAB receptor knock-out tissue. In conclusion, this study identifies tissue-specific mGlu1-associated protein clusters including KCTD12 at Purkinje cell synapses.

Long-term depression–inductive stimulation causes long-term potentiation in mouse Purkinje cells with a mutant thyroid hormone receptor

Thyroid hormones (THs) regulate gene expression by binding to nuclear TH receptors (TRs) in the cell. THs are indispensable for brain development. However, we have little knowledge about how congenital hypothyroidism in neurons affects functions of the central nervous system in adulthood. Here, we report specific TH effects on functional development of the cerebellum by using transgenic mice overexpressing a dominant-negative TR (Mf-1) specifically in cerebellar Purkinje cells (PCs). Adult Mf-1 mice displayed impairments in motor coordination and motor learning. Surprisingly, long-term depression (LTD)-inductive stimulation caused long-term potentiation (LTP) at parallel fiber (PF)-PC synapses in adult Mf-1 mice, although there was no abnormality in morphology or basal properties of PF-PC synapses. The LTP phenotype was turned to LTD in Mf-1 mice when the inductive stimulation was applied in an extracellular high-Ca2+ condition. Confocal calcium imaging revealed that dendritic Ca2+ elevation evoked by LTD-inductive stimulation is significantly reduced in Mf-1 PCs but not by PC depolarization only. Single PC messenger RNA quantitative analysis showed reduced expression of SERCA2 and IP3 receptor type 1 in Mf-1 PCs, which are essential for mGluR1-mediated internal calcium release from endoplasmic reticulum in cerebellar PCs. These abnormal changes were not observed in adult-onset PC-specific TH deficiency mice created by adeno-associated virus vectors. Thus, we propose the importance of TH action during neural development in establishing proper cerebellar function in adulthood, independent of its morphology. The present study gives insight into the cellular and molecular mechanisms underlying congenital hypothyroidism-induced dysfunctions of central nervous system and cerebellum.

MGluR5 Is Substitutable for mGluR1 in Cerebellar Purkinje Cells for Motor Coordination, Developmental Synapse Elimination, and Motor Learning.

Group I metabotropic glutamate receptors (mGluRs) include mGluR1 and mGluR5, which are coupled to the Gq family of heterotrimeric G-proteins and readily activated by their selective agonist 3,5-dihydroxyphenilglycine (DHPG). mGluR1 and mGluR5 exhibit nearly complementary distributions spatially or temporally in the central nervous system (CNS). In adult cerebellar Purkinje cells (PCs), mGluR1 is a dominant group I mGluR and mGluR5 is undetectable. mGluR1 expression increases substantially during the first three weeks of postnatal development and remains high throughout adulthood. On the other hand, mGluR5 expression is observed during the first two postnatal weeks and then decreases. However, functional differences between mGluR1 and mGluR5 in the CNS remains to be elucidated. To address this issue, we generated “mGluR5-rescue” mice in which mGluR5 is specifically expressed in PCs in global mGluR1-knockout (KO) mice. mGluR5-rescue mice exhibited apparently normal motor coordination, developmental elimination of redundant climbing fiber (CF)-PC synapses, and delay eyeblink conditioning, which were severely impaired in mGluR1-KO mice. We concluded that mGluR5 is functionally comparable with mGluR1 in cerebellar PCs.

Topographical distance between presynaptic Ca2+ channels and exocytotic Ca2+ sensors contributes to differential facilitatory actions of roscovitine on neurotransmitter release at cerebellar glutamatergic and GABAergic synapses.

Calcium influx into presynaptic terminals through voltage-gated Ca2+ channels triggers univesicular or multivesicular release of neurotransmitters depending on the characteristics of the release machinery. However, the mechanisms underlying multivesicular release (MVR) and its regulation remain unclear. Previous studies showed that in rat cerebellum, the cyclin-dependent kinase inhibitor roscovitine profoundly increases excitatory postsynaptic current (EPSC) amplitudes at granule cell (GC)-Purkinje cell (PC) synapses by enhancing the MVR of glutamate. This compound can also moderately augment the amplitude and prolong the decay time of inhibitory postsynaptic currents (IPSCs) at molecular layer interneuron (MLI)-PC synapses via MVR enhancement and GABA spillover, thus allowing for persistent activation of perisynaptic GABA receptors. The enhanced MVR may depend on the driving force for Cav 2.1 channel-mediated Ca2+ influx. To determine whether the distinct spatiotemporal dynamics of presynaptic Ca2+ influence MVR, we compared the effects of slow and fast Ca2+ chelators, that is, EGTA and BAPTA, respectively, on roscovitine-induced actions at GC-PC and MLI-PC synapses. Membrane-permeable EGTA-AM decreased GC-PC EPSC and MLI-PC IPSC amplitudes to a similar extent but suppressed the roscovitine-induced enhancement of EPSCs. In contrast, BAPTA-AM attenuated the effects of roscovitine on IPSCs. These results suggest that roscovitine augmented glutamate release by activating the release machinery located distally from the Cav 2.1 channel clusters, while it enhanced GABA release in a manner less dependent on those at distal sites. Therefore, the spatial relationships among Ca2+ channels, buffers, and sensors are critical determinants of the differential facilitatory actions of roscovitine on glutamatergic and GABAergic synapses in the cerebellar cortex.

Anatomy embroiders function in Purkinje cells

The anatomy of the human cerebellar Purkinje cell, as captured by Ramón y Cajal in a drawing, inspired the spectacular cover embroidery by Lisa Stoneman (Roanoke College, Roanoke, VA, USA) in the October issue of The Lancet Neurology. 1 Mehta AR Abbott CM Chandran S Haley JE The Cajal Embroidery Project: celebrating neuroscience. Lancet Neurol. 2020; 19: 979 Summary Full Text Full Text PDF Scopus (6) Google Scholar Like contemporary confocal microscopy images do, this anatomical representation also raises fascinating questions about the function served by the complex array of dendrites in this cell type. Each Purkinje cell, in rodents, receives about 174 000 synaptic inputs from the parallel fibre axons of the far tinier cerebellar granule cells, along with a single powerful input from the climbing fibre arising from the inferior olivary nucleus. 2 Howarth C Gleeson P Attwell D Updated energy budgets for neural computation in the neocortex and cerebellum. J Cereb Blood Flow Metab. 2012; 32: 1222-1232 Crossref PubMed Scopus (337) Google Scholar Even though a large fraction of those synapses can become inactive with learning, 3 Isope P Barbour B Properties of unitary granule cell→Purkinje cell synapses in adult rat cerebellar slices. J Neurosci. 2002; 22: 9668-9678 Crossref PubMed Google Scholar it is remarkable that the information processed from such a large number of inputs, which are even more numerous in human beings, is ultimately channelled down a single inhibitory axon to the deep cerebellar nuclei, as denoted by the label a in Cajal's original 1894 drawing (appendix).

Introduction of synaptotagmin 7 promotes facilitation at the climbing fiber to Purkinje cell synapse.

SUMMARYSynaptotagmin 7 (Syt7) is a high-affinity calcium sensor that is implicated in multiple aspects of synaptic transmission. Here, we study the influence of Syt7 on the climbing fiber (CF) to Purkinje cell (PC) synapse. We find that small facilitation and prominent calcium-dependent recovery from depression at this synapse do not rely on Syt7 and that Syt7 is not normally present in CFs. We expressed Syt7 in CFs to assess the consequences of introducing Syt7 to a synapse that normally lacks Syt7. Syt7 expression does not promote asynchronous release or accelerate recovery from depression. Syt7 decreases the excitatory postsynaptic current (EPSC) magnitude, consistent with a decrease in the initial probability of release (PR). Syt7 also increases synaptic facilitation to such a large extent that it could not arise solely as an indirect consequence of decreased PR. Thus, the primary consequence of Syt7 expression in CFs, which normally lack Syt7, is to promote synaptic facilitation.

Stochastic reaction-diffusion modeling of calcium dynamics in 3D dendritic spines of Purkinje cells

Calcium (Ca2+) is a second messenger assumed to control changes in synaptic strength in the form of both long-term depression and long-term potentiation at Purkinje cell dendritic spine synapses via inositol trisphosphate (IP3)-induced Ca2+ release. These Ca2+ transients happen in response to stimuli from parallel fibers (PFs) from granule cells and climbing fibers (CFs) from the inferior olivary nucleus. These events occur at low numbers of free Ca2+, requiring stochastic single-particle methods when modeling them. We use the stochastic particle simulation program MCell to simulate Ca2+ transients within a three-dimensional Purkinje cell dendritic spine. The model spine includes the endoplasmic reticulum, several Ca2+ transporters, and endogenous buffer molecules. Our simulations successfully reproduce properties of Ca2+ transients in different dynamical situations. We test two different models of the IP3 receptor (IP3R). The model with nonlinear concentration response of binding of activating Ca2+ reproduces experimental results better than the model with linear response because of the filtering of noise. Our results also suggest that Ca2+-dependent inhibition of the IP3R needs to be slow to reproduce experimental results. Simulations suggest the experimentally observed optimal timing window of CF stimuli arises from the relative timing of CF influx of Ca2+ and IP3 production sensitizing IP3R for Ca2+-induced Ca2+ release. We also model ataxia, a loss of fine motor control assumed to be the result of malfunctioning information transmission at the granule to Purkinje cell synapse, resulting in a decrease or loss of Ca2+ transients. Finally, we propose possible ways of recovering Ca2+ transients under ataxia.

The effect of reactive Bergmann glia on short-term synaptic plasticity in cerebellar neurodegenerative models, caused by chronic activation of ChR2 and expression of the mutant ataxin-1

Introduction. Synaptic plasticity is impaired in the early stages of a neurodegenerative process but is potentially reversible. The study of mechanisms associated with synaptic plasticity in neurodegenerative cerebellar conditions has enabled the search for potential therapeutic agents. This study aimed to investigate the effect of the astrocytic link on paired-pulse facilitation (PPF) in cerebellar cortical synapses of mice, using a set of immunohistochemical, optogenetic, and electrophysiological analysis methods. Materials and methods. Experiments were conducted on 12-week-old CD-1 mice. The model of murine cerebellar astrogliosis was created using chronic activation of light-sensitive ChR2 channels in Bergmann glia and after they expressed the mutant ataxin-1. To model astrocyte-mediated neurodegeneration, these mice were intracortically administered AVV GFAP-ChR2-mKate vector constructions with subsequent chronic 4-day light stimulation in vivo and LVV GFAP-ATXN1[Q85]-Flag without light stimulation. Mice in the control group were administered normal saline or LVV GFAP-ATXN1[Q2]-Flag. Changes in the PFF-excitatory postsynaptic currents in Purkinje cells were registered using the patch-clamp technique. Immunohistochemistry was used to examine anti-GFAP, mKate, and anti-ataxin-1 expression in the cerebellar cortex. Results. For the reactive glia in the cerebellar cortex after chronic photostimulation, increased anti-GFAP immune reactivity and morphology changes in the form of process tortuosity were common. In Purkinje cell synapses with parallel fibers in these animals, the PPF coefficient was significantly increased because of impaired glutamate reuptake and presynaptic overexcitation with this neuromediator. However, photoactivation of reactive Bergmann glia led to a sharp slowing down of the glutamate-glutamine cycle and glutamate pool depletion in the presynapse, with a subsequent gradual reduction in the PPF coefficient. Such pathological mechanisms were found in the neurodegenerative model with selective damage to Bergmann glia by the mutant ataxin-1. Conclusion. Astrocytes affect short-term synaptic plasticity such as PPF. In cerebellar astrogliosis, the PPF disturbance is multilevel: the high baseline level of PPF is significantly reduced after Bergmann glial activation, which is related to impaired glutamate reuptake by reactive glial cells. For citation: Shuvaev А.N., Belozor О.S., Mozjei O.I., Yakovleva D.A., Shuvaev А.N., Smolnikova М.V., Pozhilenkova Е.A., Каsparov S., Salmin V.V., Salmina А.B. [The effect of reactive Bergmann glia on short-term synaptic plasticity in cerebellar neurodegenerative models, caused by chronic activation of ChR2 and expression of the mutant ataxin-1]. Annals of clinical and experimental neurology. 2021; 15(1): 51–58. (In Russ.) https://doi.org/10.25692/ACEN.2021.1.6

A Cerebellar Computational Mechanism for Delay Conditioning at Precise Time Intervals.

The cerebellum is known to have an important role in sensing and execution of precise time intervals, but the mechanism by which arbitrary time intervals can be recognized and replicated with high precision is unknown. We propose a computational model in which precise time intervals can be identified from the pattern of individual spike activity in a population of parallel fibers in the cerebellar cortex. The model depends on the presence of repeatable sequences of spikes in response to conditioned stimulus input. We emulate granule cells using a population of Izhikevich neuron approximations driven by random but repeatable mossy fiber input. We emulate long-term depression (LTD) and long-term potentiation (LTP) synaptic plasticity at the parallel fiber to Purkinje cell synapse. We simulate a delay conditioning paradigm with a conditioned stimulus (CS) presented to the mossy fibers and an unconditioned stimulus (US) some time later issued to the Purkinje cells as a teaching signal. We show that Purkinje cells rapidly adapt to decrease firing probability following onset of the CS only at the interval for which the US had occurred. We suggest that detection of replicable spike patterns provides an accurate and easily learned timing structure that could be an important mechanism for behaviors that require identification and production of precise time intervals.

SAT-708 The Effect of Soybean Isoflavones in Developing Cerebellum

ENDO 2020The effect of soybean isoflavones in developing cerebellumThyroid hormone (TH) receptor (TR) and estrogen receptor (ER) play crucial roles in cerebellar development. TR and ER are involved in Purkinje cells dendrite growth, spines and synapse formation. They also regulate the functional maturation, intracellular metabolism, and migration of neuron and glial. Soybean isoflavones especially genistein, daidzein, and daidzein metabolite, S-equol were known to exert their action through TR, ER, and GPR30, that is a G-protein-coupled ER. However, the mechanisms of soybean isoflavone action on cerebellar development and function have not yet been extensively studied. We evaluated the effects of soybean isoflavone, such as genistein, daidzein, and S-equol, using mouse primary cerebellar culture, astrocyte-enriched culture, and C6 clonal cells. Soybean isoflavone augmented TH- or estradiol (E2)-mediated dendrite arborization of Purkinje cells. Such augmentation was suppressed by G15, a selective G-protein coupled ER (GPR30) antagonist, and ICI 182.780, an antagonist for ERs in both cultures. It also increased mRNA expression level of TH-responsive genes including Mbp, Bdnf, Rc3, Ntf3, Camk2b, and Hr. Moreover, genistein and daidazein also increased mRNA expression level of Syn1, Syp and Psd95 that are involved in synaptic plasticity.On the other hand, in astrocytes, soybean isoflavone activated cell migration and F-actin rearrangements. Such effects were suppressed by G15, but not by ICI 182.780. Knockdown of GPR30 by RNAi also suppressed the cells migration. Protein expression levels of p-Akt (Ser473), p-Rac1/cdc42 (Ser71), RhoA, Rac1/2/3, and cdc42 also increased by soybean isoflavone. Co-exposure with Rhosin HCl, a selective RhoA inhibitor, reduced the cells migration and formation of stress fibers. These findings indicate that sobybean isoflavone may affect cerebellar development by acting to both neurons and astrocytes through several signaling pathways, including TR, ER, and GPR30.Keywords:EDC, ER, TR, GPR30, Neuron, Astrocyte

AMPAR Auxiliary Protein SHISA6 Facilitates Purkinje Cell Synaptic Excitability and Procedural Memory Formation

SummaryThe majority of excitatory postsynaptic currents in the brain are gated through AMPA-type glutamate receptors, the kinetics and trafficking of which can be modulated by auxiliary proteins. It remains to be elucidated whether and how auxiliary proteins can modulate synaptic function to contribute to procedural memory formation. In this study, we report that the AMPA-type glutamate receptor (AMPAR) auxiliary protein SHISA6 (CKAMP52) is expressed in cerebellar Purkinje cells, where it co-localizes with GluA2-containing AMPARs. The absence of SHISA6 in Purkinje cells results in severe impairments in the adaptation of the vestibulo-ocular reflex and eyeblink conditioning. The physiological abnormalities include decreased presence of AMPARs in synaptosomes, impaired excitatory transmission, increased deactivation of AMPA receptors, and reduced induction of long-term potentiation at Purkinje cell synapses. Our data indicate that Purkinje cells require SHISA6-dependent modification of AMPAR function in order to facilitate cerebellar, procedural memory formation.

Differential synapse density between Purkinje cell dendritic spine and parallel fiber varicosity in the rat cerebellum among the phylogenic lobules

The cerebellum is a region of the brain that plays an important role in motor control. It is classified phylogenetically into archicerebellum, paleocerebellum and neocerebellum. The Purkinje cells are lined in a row called Purkinje cell layer and it has a unique dendritic branches with many spines.The previous study reported that there is a difference of synapse density according to the lobules based on large two-dimensional data. However, recent study with high voltage electron microscopy showed there was no differences in dendritic spine density of the Purkinje cell according to its phylogenetic lobule. We analyzed Purkinje cell density in the II, VI and X lobules by stereological modules and synaptic density was estimated by double disector based on Purkinje cell density in the molecular layer of each lobule.The results showed that there was significant difference in the Purkinje cell density and synapse number according to their phylogenetic lobules. The number of Purkinje cell in a given volume was larger in the archicerebellum, but synapse density was higher in the neocerebellum.These data suggest that cellular and synaptic organization of the Purkinje cell is different according to their phylogenetic background.

A Light-Controlled Allosteric Modulator Unveils a Role for mGlu4 Receptors During Early Stages of Ischemia in the Rodent Cerebellar Cortex.

Metabotropic glutamate receptors (mGlus) are G Protein coupled-receptors that modulate synaptic transmission and plasticity in the central nervous system. Some act as autoreceptors to control neurotransmitter release at excitatory synapses and have become attractive targets for drug therapy to treat certain neurological disorders. However, the high degree of sequence conservation around the glutamate binding site makes the development of subtype-specific orthosteric ligands difficult to achieve. This problem can be circumvented by designing molecules that target specific less well conserved allosteric sites. One such allosteric drug, the photo-switchable compound OptoGluNAM4.1, has been recently employed to reversibly inhibit the activity of metabotropic glutamate 4 (mGlu4) receptors in cell cultures and in vivo. We studied OptoGluNAM4.1 as a negative modulator of neurotransmission in rodent cerebellar slices at the parallel fiber – Purkinje cell synapse. Our data show that OptoGluNAM4.1 antagonizes pharmacological activation of mGlu4 receptors in a fully reversible and photo-controllable manner. In addition, for the first time, this new allosteric modulator allowed us to demonstrate that, in brain slices from the rodent cerebellar cortex, mGlu4 receptors are endogenously activated in excitotoxic conditions, such as the early phases of simulated cerebellar ischemia, which is associated with elevated levels of extracellular glutamate. These findings support OptoGluNAM4.1 as a promising new tool for unraveling the role of mGlu4 receptors in the central nervous system in physio-pathological conditions.

Purkinje Cell Signaling Deficits in Animal Models of Ataxia.

Purkinje cell (PC) dysfunction or degeneration is the most frequent finding in animal models with ataxic symptoms. Mutations affecting intrinsic membrane properties can lead to ataxia by altering the firing rate of PCs or their firing pattern. However, the relationship between specific firing alterations and motor symptoms is not yet clear, and in some cases PC dysfunction precedes the onset of ataxic signs. Moreover, a great variety of ionic and synaptic mechanisms can affect PC signaling, resulting in different features of motor dysfunction. Mutations affecting Na+ channels (NaV1.1, NaV1.6, NaVβ4, Fgf14 or Rer1) reduce the firing rate of PCs, mainly via an impairment of the Na+ resurgent current. Mutations that reduce Kv3 currents limit the firing rate frequency range. Mutations of Kv1 channels act mainly on inhibitory interneurons, generating excessive GABAergic signaling onto PCs, resulting in episodic ataxia. Kv4.3 mutations are responsible for a complex syndrome with several neurologic dysfunctions including ataxia. Mutations of either Cav or BK channels have similar consequences, consisting in a disruption of the firing pattern of PCs, with loss of precision, leading to ataxia. Another category of pathogenic mechanisms of ataxia regards alterations of synaptic signals arriving at the PC. At the parallel fiber (PF)-PC synapse, mutations of glutamate delta-2 (GluD2) or its ligand Crbl1 are responsible for the loss of synaptic contacts, abolishment of long-term depression (LTD) and motor deficits. At the same synapse, a correct function of metabotropic glutamate receptor 1 (mGlu1) receptors is necessary to avoid ataxia. Failure of climbing fiber (CF) maturation and establishment of PC mono-innervation occurs in a great number of mutant mice, including mGlu1 and its transduction pathway, GluD2, semaphorins and their receptors. All these models have in common the alteration of PC output signals, due to a variety of mechanisms affecting incoming synaptic signals or the way they are processed by the repertoire of ionic channels responsible for intrinsic membrane properties. Although the PC is a final common pathway of ataxia, the link between specific firing alterations and neurologic symptoms has not yet been systematically studied and the alterations of the cerebellar contribution to motor signals are still unknown.

Protein kinase N1 critically regulates cerebellar development and long-term function.

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.

Inositol hexakisphosphate kinase‐2 in cerebellar granule cells acts through protein 4.1N to regulate Purkinje cell morphology and motor coordination

Inositol hexakisphosphate kinases (IP6Ks) are responsible for the synthesis of the energy‐rich Inositol pyrophosphates (PP‐IPs) in mammals and regulate cellular functions including chemotaxis, telomere length, endocytic trafficking, exocytosis as well as apoptosis. Among such pyrophosphates, diphosphoinositol pentakisphosphate (IP7) and bis‐diphosphoinositol tetrakisphosphate (IP8) have been extensively characterized. IP7 is produced in mammals by a family of inositol hexakisphosphate kinases, namely IP6K1, IP6K2 and IP6K3, which have distinct biological functions. Of these, Inositol hexakisphosphate kinase‐2 (IP6K2), has been identified as a pro‐apoptotic factor with the ability to sensitize cells to apoptosis. To identify the protein interactome of IP6K2, we subjected wild‐type mouse brain tissue lysates to anti‐IP6K2 antibody‐mediated coimmuno‐precipitation followed by Liquid Chromatography Tandem Mass‐Spectrometry (LC‐MS/MS). This screen revealed robust binding of protein 4.1N with IP6K2. 4.1N is believed to confer stability and plasticity to the neuronal membrane via interactions with multiple binding partners, including the spectrin‐actin‐based cytoskeleton, integral membrane channels and receptors as well as membrane‐associated guanylate kinases. To further assess the specificity of the observed interaction between IP6K2 and 4.1N, we separately immuno‐precipitated IP6K1 and IP6K3 from brain tissue lysates of mice and failed to detect any association with the 4.1N protein. Thus, the interaction of 4.1N is evidently specific to the IP6K2 form of inositol hexakisphosphate kinases. Moreover, nuclear translocation of 4.1N, which is required for its principal functions, was dependent on IP6K2. We also found that IP6K2 and 4.1N are expressed in the granule cells and that their interaction regulate Purkinje cell morphology and synapse in the cerebellum. This was determined through Golgi staining, double immunofluorescence staining and electron microscopy. Deletion of IP6K2 in mice led to substantial defects in synaptic influences of granule cells upon Purkinje cells. Rotarod, open field test and Gait analysis also revealed the defects of motor coordination in IP6K2 knockout mice indicating the functional consequences of these interaction. Overall, our study establishes the functional implication of the robust association between IP6K2‐4.1N in the brain.Support or Funding InformationThe present funding capabilities and strategies of our lab do not have provisions for covering the defined expenditure related to the current scientific meeting. Thus, to enrich my research skills and knowledge in neurophysiology as well as inositol phosphates, I would request possible financial support through the Postdoctoral Travel Award funds to help me attend this meeting.This would allow me to share my work and ideas with young scientists as well as researchers from US and other parts of the world thus helping me to get my research ideas as well as work, evaluated and advised on, by the best in the worldI would therefore entreat your good offices to consider my present submission for possible financial aid to help me attend the present meeting on Experimental Biology 2018This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.