Mouse Cerebellum Research Articles

Synthetic α-Tocopherol, Compared with Natural α-Tocopherol, Downregulates Myelin Genes in Cerebella of Adolescent Ttpa-null Mice

Background:Vitamin E (α-tocopherol; αα-T) deficiency causes spinocerebellar ataxia. α-T supplementation improves neurological symptoms, but little is known about the differential bioactivities of natural versus synthetic α-T during early life. Objectives:We assessed the effects of dietary α-T dose and source on tissue α-T accumulation and gene expression in adolescent α-Tocopherol transfer protein-null (Ttpa–/–) mice. Methods:Three-week-old male Ttpa–/– mice (n = 7/group) were fed 1 of 4 AIN-93Gbased diets for 4 wk: vitamin Edeficient (VED; below α-T limit of detection); natural α-T, 600 mg/kg diet (NAT); synthetic α-T, 816 mg/kg diet (SYN); orhigh synthetic α-T, 1200 mg/kg diet (HSYN). Male Ttpa+/+ littermates fed AIN-93G [75 mg synthetic α-T (CON)] servedas controls (n = 7). At 7 wk of age, tissue α-T concentrations and stereoisomer profiles were measured for all groups. Results:Ttpa–/– mice fed VED had undetectable brain α-T concentrations. Cerebral cortex α-T concentrations weregreater in Ttpa–/– mice fed NAT (9.1 ± 0.7 nmol/g), SYN (10.8 ± 1.0 nmol/g), and HSYN (13.9 ± 1.6 nmol/g) compared withthe VED group but were significantly lower than in Ttpa+/+ mice fed CON (24.6 ± 1.2 nmol/g) (P < 0.001). RRR-α-T wasthe predominant stereoisomer in brains of Ttpa+/+ mice (~40%) and Ttpa–/– mice fed NAT (~94%). α-T stereoisomercomposition was similar in brains of Ttpa–/– mice fed SYN and HSYN (2R: ~53%; 2S: ~47%). Very few of the 16,774genes measured were differentially expressed. However, compared with the NAT diet, HSYN significantly downregulated20 myelin genes, including 2 transcription factors: SRY-box transcription factor 10 (Sox10) and myelin regulatory factor(Myrf), and several downstream target genes (false discovery rate <0.05). Conclusions:High-dose synthetic α-T compared with natural α-T alters myelin gene expression in the adolescent mouse cerebellum, which could lead to morphological and functional abnormalities later in life.

Phospholipase C β3 is Required for Climbing Fiber Synapse Elimination in Aldolase C-positive Compartments of the Developing Mouse Cerebellum

In the cerebellum of neonatal mice, multiple climbing fibers (CFs) form excitatory synapses on each Purkinje cell (PC). Only one CF is strengthened in each PC from postnatal day 3 (P3) to P7, whereas the other weaker CFs are eliminated progressively from ∼P7 to ∼P11 (early phase of CF elimination) and from ∼P12 to ∼P17 (late phase of CF elimination). Type 1 metabotropic glutamate receptor (mGluR1) triggers a canonical pathway in PCs for the late phase of CF elimination. Among downstream signaling molecules of mGluR1, phospholipase C β3 (PLCβ3) and β4 (PLCβ4) are expressed complementarily in PCs of aldolase C (Aldoc)-positive (+) and Aldoc-negative (−) cerebellar compartments, respectively. PLCβ4 is reported to mediate the late phase of CF elimination in the anterior half of the cerebellar vermis which corresponds to the Aldoc (−) region. However, roles of PLCβ3 and Aldoc in CF synapse elimination are unknown. Here, we investigated CF innervation of PCs in Aldoc-tdTomato knock-in mice that underwent lentivirus-mediated knockdown (KD) of PLCβ3 in PCs during postnatal development. By recording CF-mediated excitatory postsynaptic currents from PCs and immunostaining CF synaptic terminals, we found that significantly higher percentage of PCs with PLCβ3-KD remained multiply innervated by CFs in Aldoc (+) compartments after P12, which was accompanied by impaired elimination of somatic CF synapses and reduced dendritic CF translocation. In contrast, deletion of Aldoc had no effect on CF synapse elimination. These results suggest that PLCβ3 is required for the late phase of CF elimination in Aldoc (+) PCs.

Alteration of the Dopamine Receptors’ Expression in the Cerebellum of the Lysosomal Acid Phosphatase 2 Mutant (Naked–Ataxia (NAX)) Mouse

A spontaneous mutation in the lysosomal acid phosphatase (Acp2) enzyme (nax: naked–ataxia) in experimental mice results in delayed hair appearance and severe cytoarchitectural impairments of the cerebellum, such as a Purkinje cell (PC) migration defect. In our previous investigation, our team showed that Acp2 expression plans a significant role in cerebellar development. On the other hand, the dopaminergic system is also a player in central nervous system (CNS) development, including cerebellar structure and function. In the current investigation, we have explored how Acp2 can be involved in the regulation of the dopaminergic pathway in the cerebellum via the regulation of dopamine receptor expression and patterning. We provided evidence about the distribution of different dopamine receptors in the developing cerebellum by comparing the expression of dopamine receptors on postnatal days (P) 5 and 17 between nax mice and wild–type (wt) littermates. To this aim, immunohistochemistry and Western blot analysis were conducted using five antibodies against dopamine receptors (DRD1, –2, –3, –4, and –5) accompanied by RNAseq data. Our results revealed that DRD1, –3, and –4 gene expressions significantly increased in nax cerebella but not in wt, while gene expressions of all 5 receptors were evident in PCs of both wt and nax cerebella. DRD3 was strongly expressed in the PCs’ somata and cerebellar nuclei neurons at P17 in nax mice, which was comparable to the expression levels in the cerebella of wt littermates. In addition, DRD3 was expressed in scattered cells in a granular layer reminiscent of Golgi cells and was observed in the wt cerebella but not in nax mice. DRD4 was expressed in a subset of PCs and appeared to align with the unique parasagittal stripes pattern. This study contributes to our understanding of alterations in the expression pattern of DRDs in the cerebellum of nax mice in comparison to their wt littermates, and it highlights the role of Acp2 in regulating the dopaminergic system.

Purification of Prominin-1+ Stem Cells from Postnatal Mouse Cerebellum.

Most cerebellar neurons arise from two embryonic stem niches: a rhombic lip niche, which generates all the cerebellar excitatory glutamatergic neurons, and a ventricular zone niche, which generates the inhibitory GABAergic Purkinje cells, which are neurons that constitute the deep cerebellar nuclei and Bergman glia. Recently, a third stem cell niche has been described that arises as a secondary germinal zone from the ventricular zone niche. The cells of this niche are defined by the cell surface marker prominin-1 and are localized to the developing white matter of the postnatal cerebellum. This niche accounts for the late born molecular layer GABAergic interneurons along with postnatally generated cerebellar astrocytes. In addition to their developmental role, this niche is gaining translational importance in regards to its involvement in neurodegeneration and tumorigenesis. The biology of these cells has been difficult to decipher because of a lack of efficient techniques for their purification. Demonstrated here are efficient methods to purify, culture, and differentiate these postnatal cerebellar stem cells.

Dual-Targeted Autoimmune Sword in Fatal Epilepsy: Patient's glutamate receptor AMPA GluR3B peptide autoimmune antibodies bind, induce Reactive Oxygen Species (ROS) in, and kill both human neural cells and T cells

Nodding Syndrome (NS) is a fatal pediatric epilepsy of unknown etiology, accompanied by multiple neurological impairments, and associated with Onchocerca volvulus (Ov), malnutrition, war-induced trauma, and other insults. NS patients have neuroinflammation, and ~50% have cross-reactive Ov/Leiomodin-1 neurotoxic autoimmune antibodies. ResultsStudying 30 South Sudanese NS patients and a similar number of healthy subjects from the same geographical region, revealed autoimmune antibodies to 3 extracellular peptides of ionotropic glutamate receptors in NS patients: AMPA-GluR3B peptide antibodies (86%), NMDA-NR1 peptide antibodies (77%) and NMDA-NR2 peptide antibodies (87%) (in either 1:10, 1:100 or 1:1000 serum dilution). In contrast, NS patients did not have 26 other well-known autoantibodies that target the nervous system in several autoimmune-mediated neurological diseases. We demonstrated high expression of both AMPA-GluR3 and NMDA-NR1 in human neural cells, and also in normal human CD3+ T cells of both helper CD4+ and cytotoxic CD8+ types. Patient's GluR3B peptide antibodies were affinity-purified, and by themselves precipitated short 70 kDa neuronal GluR3. NS patient's affinity-purified GluR3B peptide antibodies also bound to, induced Reactive Oxygen Species (ROS) in, and killed both human neural cells and T cells within 1-2 hours only. NS patient's purified IgGs, or serum (1:10 or 1:30), induced similar effects. In vivo video EEG experiments in normal mice, revealed that when NS patient's purified IgGs were released continuously (24/7 for 1 week) in normal mouse brain, they induced all the following: 1.Seizures, 2. Cerebellar Purkinje cell loss, 3. Degeneration in the hippocampus and cerebral cortex, and 4. Elevation of CD3+ T cells, and of activated Mac-2+microglia and GFAP+astrocytes in both the gray and white matter of the cerebral cortex, hippocampus, corpus calossum and cerebellum of mice. NS patient's serum cytokines: IL-1β, IL-2, IL-6, IL-8, TNFα, IFNγ, are reduced by 85–99% compared to healthy subjects, suggesting severe immunodeficiency in NS patients. This suspected immunodeficiency could be caused by combined effects of the: 1. Chronic Ov infection, 2. Malnutrition, 3. Killing of NS patient's T cells by patient's own GluR3B peptide autoimmune antibodies (alike the killing of normal human T cells by the NS patient's GluR3B peptide antibodies found herein in vitro). ConclusionsRegardless of NS etiology, NS patients suffer from ‘Dual-targeted Autoimmune Sword’: autoimmune AMPA GluR3B peptide antibodies that bind, induce ROS in, and kill both neural cells and T cells. These neurotoxic and immunotoxic GluR3B peptide autoimmune antibodies, and also NS patient's NMDA-NR1/NR2A and Ov/Leiomodin-1 autoimmune antibodies, must be silenced or removed. Moreover, the findings of this study are relevant not only to NS, but also to many more patients with other types of epilepsy, which have GluR3B peptide antibodies in serum and/or CSF. This claim is based on the following facts: 1. The GluR3 subunit is expressed in neural cells in crucial brains regions, in motor neurons in the spinal cord, and also in other cells in the body, among them T cells of the immune system, 2. The GluR3 subunit has diverse neurophysiological role, and its deletion or abnormal function can: disrupt oscillatory networks of both sleep and breathing, impair motor coordination and exploratory activity, and increase the susceptibility to generate seizures, 3. GluR3B peptide antibodies were found so far in ~27% of >300 epilepsy patients worldwide, which suffer from various other types of severe, intractable and enigmatic epilepsy, and which turned out to be 'Autoimmune Epilepsy'. Furthermore, the findings of this study could be relevant to different neurological diseases besides epilepsy, since other neurotransmitter-receptors autoantibodies are present in other neurological and psychiatric diseases, e.g. autoimmune antibodies against other GluRs, Dopamine receptors, GABA receptors, Acetylcholine receptors and others. These neurotransmitter-receptors autoimmune autoantibodies might also act as ‘Dual-targeted Autoimmune Sword’ and damage both neural cells and T cells (as the AMPA-GluR3B peptide antibodies induced in the present study), since T cells, alike neural cells, express most if not all these neurotransmitter receptors, and respond functionally to the respective neurotransmitters - a scientific and clinical topic we coined 'Nerve-Driven Immunity'.

The impact of rattlesnake venom on mice cerebellum proteomics points to synaptic inhibition and tissue damage

Snake envenomation is responsible for more than 130,000 deaths worldwide. In Brazil, the Crotalus rattlesnake is responsible for the second largest number of accidental snake bites in the country. Although there are many descriptions of the clinical and biochemical effects of Crotalus envenoming, there are few works describing the molecular events in the central nervous system of an organism due to envenomation. In this study, we analyzed the proteomic effect of Crotalus durissus terrificus snake venom on mice cerebellums. To monitor the envenomation over time, changes in the protein abundance were evaluated at 1 h, 6 h, 12 h and 24 h after venom injection by mass spectrometry. The analysis of the variation of over 4600 identified proteins over time showed a reduction in components of inhibitory synapse signaling, oxidative stress, and maintenance of neuronal cells, which paralleled increasing tissue damage and apoptosis factors. These analyses revealed the potential protein targets of the C. d. terrificus venom on the murine cerebellum, showing new aspects of the snake envenomation effect. These data may contribute to new therapeutic approaches (i.e., approaches directed at protein targets affected by the envenomation) on the treatment of envenomation by the neurotoxic C. d. terrificus snake venom. SignificanceSnakebites are a neglected global health problem that affects mostly rural and tropical areas of developing countries. It is estimated that over 5.4 million people are bitten by snakes each year, from which 2.7 million people are bitten by venomous snakes, resulting in disabilities such as amputations and in some cases leading to death.The C. d. terrificus snake is the most lethal snake in Brazil. Studying the molecular changes upon envenomation in a specific tissue may lead to a better understanding of the envenomation process by C. d. terrificus snakebites.

Identification of RNA Exosome Cofactors in Neuronal Cells to Probe Disease Mechanism

The RNA exosome, a 10‐subunit complex that mediates both RNA processing and degradation, plays a critical role in defining cellular expression profiles. This complex is ubiquitously expressed, essential, and critical for fundamental cellular functions, such as ribosomal RNA processing. Recent studies have linked mutations in genes encoding multiple subunits of the complex to tissue‐specific human disease. For example, missense mutations in the human EXOSC3 gene, which encodes an RNA exosome subunit, cause Pontocerebellar Hypoplasia type 1b (PCH1b), a disease characterized by atrophy of the pons and cerebellum. The missense mutations encode single amino acid changes in conserved regions of the EXOSC3 protein. How these amino acid substitutions confer tissue‐specific phenotypes is not known. One possible mechanism underlying the distinct disease phenotypes could be a decrease in the interaction of the RNA exosome complex with cofactors that confer specificity for RNA targets. However, most studies that identify and characterize RNA exosome cofactors have been carried out in budding yeast and thus, do not provide insight into whether tissue‐specific cofactors could exist. Our studies use immunoprecipitation from neuronal cell culture (N2A) and relevant mouse tissues to define RNA exosome cofactors. Biochemical experiments that employ cultured N2A cells were used to identify RNA exosome‐interacting proteins in both the nucleus and the cytoplasm. Preliminary results from this analysis reveal an association between the RNA exosome and a large complex of cytoplasmic tRNA ligase enzymes, which could link defects in tRNA maturation to disease pathology. We are extending these studies to explore the possibility of tissue‐specific cofactors by immunoprecipitating EXOSC3 and analyzing co‐purifying proteins from the mouse cerebellum (affected in disease) and cortex (unaffected). To complement these approaches to identify tissue‐specific RNA exosome cofactors, we are also exploring whether amino acid changes that are linked to disease alter cofactor interactions. These studies, which are being performed in N2A cells, have identified cofactors that show altered interactions with the EXOSC3 variants present in disease. These studies will provide insight into both the functional consequences of amino acid substitutions in the RNA exosome that cause disease and the role of cofactors in conferring RNA target specificity.

Role of Inositol Hexakisphosphate Kinase‐2 (IP6K2) in regulating mitochondrial brain functions

Inositol hexakisphosphate kinase (IP6K) isoform ‐ IP6K2, selectively controls cellular apoptosis. To explore the possible role of IP6K2 in degenerative brain damage, we elucidated its protein interactome in mouse brain to identify its functional partners. IP6K2‐immunoprecipitates from wild‐type mouse brain lysates were subjected to co‐immunoprecipitation followed by liquid chromatography mass spectrometric analysis. This revealed robust association of IP6K2 with creatine kinase‐B (CK‐B), an enzyme responsible for synthesis of phosphocreatine and ATP. Notably, both IP6K2 and CK‐B were highly expressed in the mouse cerebellum. IP6K2‐knockout mice (IP6K2‐KO) produced less phosphocreatine and ATP and generated higher levels of ROS and consequent protein oxidative damage in their cerebellum compared to wild‐type counterparts. As mitochondrial respiratory chains regulate energy metabolism/ATP production through glycolysis and TCA cycle coupled to oxidative phosphorylation and electron transfer, functional abrogation of any event that controls these processes can lead to abnormal electron transfer, increased ROS and consequent mitochondrial oxidative stress/damage and cell death as observed in the IP6K2‐KO mice. IP6K2‐KO mice also showed significant impairment of locomotive and cognitive functions. We thus propose to identify the precise metabolic events that are controlled by IP6K2‐CK‐B interaction and examine the possible causal role of oxidative stress and determine whether antioxidant treatment can restore normal brain functions. We plan to explore mitochondria‐targeted antioxidants like CoQ10/Mito Q along with derivatives of higher inositol phosphates (IP7‐product of IP6K2) in the IP6K2‐KO mice to reinforce the therapeutic potential of such anti‐oxidative treatment for the clinical management of diseases associated with mitochondrial dysfunction of the brain. Overall, our proposed study would help to establish the precise role of IP6K2‐CK‐B interaction in controlling cellular degeneration in the brain.

Pharmacological inhibition of soluble epoxide hydrolase alleviates chronic neuropathic pain induced anhedonia symptoms: differential role of AhR and TSPO signaling

Patients who suffer from chronic pain have a higher incidence to develop depression, which needs effective treatments. TPPU, a soluble epoxide hydrolase (sEH) inhibitor, was reported to have analgesia and antidepressant effects in animal models, suggesting its potential improvement in this comorbidity. Here, we sought to investigate: (1) the occurrence of anhedonia symptoms in mice with chronic neuropathic pain and the sEH protein expression in different tissues, including central nervous system and peripheral tissues; (2) the effects of TPPU on nociceptive and anhedonia behaviors in anhedonia susceptible mice, and (3) the involvement of Aryl hydrocarbon receptor (AhR) signaling and translocator protein (TSPO) signaling as possible targets for this comorbidity. Spared nerve injury (SNI) was constructed to mimic chronic neuropathic pain (CNP) model, and that mice in SNI group were classified into chronic pain with or without anhedonia phenotype via hierarchical cluster analysis of sucrose preference test (SPT) results. Protein levels of sEH were increased in medial prefrontal cortex (mPFC), hippocampus, spinal cord, liver, kidney and gut, but not in anterior cingulate cortex (ACC), nucleus accumbens (NAc), striatum, cerebellum, heart, muscle and vessel in anhedonia susceptible mice as compared with sham or anhedonia resilient mice. Chronic treatment with TPPU (3 mg/kg, orally, once per day for 7 days) improved withdraw threshold and SPT scores in anhedonia susceptible mice. Interestingly, the antidepressant, but not analgesia effect, of TPPU seems to be pharmacologically abrogated by pretreatment with AhR agonist (FICZ, orally, 100 μg/kg) via regulating inflammatory cytokines and CYP1A1 levels in the serum as well as neuroinflammation in the microglia and astrocytes. Interestingly, the classical TSPO pathway, a crucial factor in the analgesia and antidepressant effects of TPPU, failed to show any difference in subjects who were treated with drugs targeted at the AhR. Collectively, our findings indicate that TPPU may alleviate depression‐like symptoms in CNP mice through two independent pathways: (1) the direct antidepressant effects via regulating neuroinflammation mediated by AhR, and (2) the improvement of allodynia and depressive symptoms via the TSPO signaling and downstream neurosteroids. Thus, AhR provides a potential therapeutic target for treatment of depression induced by chronic pain, and strategies targeting TSPO signaling could be a vital approach to alleviate the comorbidity of chronic pain and depression.Support or Funding InformationThis work was supported , in part, by grants from the National Natural Science Foundation of China (to A.L., 81771159 and 81571047; to C.Y., 81703482)The overview of our manuscript.Figure 1

Protective Effects of Berberis vulgaris on Diazinon-Induced Brain Damage in Young Male Mice.

Diazinon is an imminent and hazardous chemical organophosphate multiplex that is generally used as a pesticide but is toxic for many species particularly vertebrates. Berberry (Berberis vulgaris L., family Berberidaceae) is a plant that flourishes in Europe and Asia that has been largely investigated for its therapeutic effects. In the present study, we evaluated the protective effects of B. vulgaris on diazinon-induced brain damage in young male mice. Twenty-one young male albino mice weighing 18±2 g were divided in three equal groups of seven mice, and treated orally with either olive oil (control), diazinon 50 mg/kg+B. vulgaris extract 200 mg/kg, or diazinon 50 mg/kg. After three weeks, cerebrum and cerebellum samples were collected for antioxidant assays. The results indicated that diazinon increased oxidative stress in the brain of mice. The glutathione content and proceedings of antioxidant enzymes, such as glutathione peroxidase, superoxide dismutase, and catalase, were significantly reduced in both the cerebellum and cerebrum of diazinon-treated mice, compared with the control group. In addition, acetylcholinesterase (AChE) activity was inhibited by exposure to this pesticide. Administration of 200 mg/kg B. vulgaris extract with diazinon significantly decreased oxidative stress indices in all experiments. The results indicated that B. vulgaris extract has protective effects against lipid peroxidation of the cerebellum and cerebrum, and in regenerating AChE activity in the brain induced by diazinon.

Hippocampal and Cerebellar Changes in Acute Restraint Stress and the Impact of Pretreatment with Ceftriaxone.

Acute restraint stress (ARS) is an unavoidable stress situation and may be encountered in different clinical situations. The aim of the current study was to investigate the effects of ARS on the hippocampus and cerebellum, assess the impact of these effects on the behavior and cognitive function, and determine whether pretreatment with ceftriaxone would attenuate the damages produced by ARS on the hippocampus and cerebellum. Four groups of male mice were included in this study: The control group, ARS group, ceftriaxone group, and ARS + ceftriaxone group. Tail suspension test, Y-maze task, and open field tests were used to assess depression, working spatial memory, and anxiety. The biochemical analyses included measurements of serum cortisol, tumor necrotic factor (TNF), interleukin-6, hippocampal expression of bone morphogenetic protein 9 (BMP9), lysosomal-associated membrane protein 1 (LAMP1), glutamate transporter 1 (GLT1), heat shock protein 90, cerebellar expression of S100 protein, glutamic acid decarboxylase (GAD), and carbon anhydrase. Histopathological examination of the brain sections was conducted on the hippocampus and cerebellum by hematoxylin and eosin stains in addition to ultrastructure evaluation using electron microscopy. Our results suggested that ceftriaxone had neuroprotective properties by attenuating the effects of ARS on the hippocampus and cerebellum in mice. This effect was demonstrated by the improvement in the cognitive and behavioral tests as well as by the preservation of the hippocampal and cerebellar architecture.

Hippocampal mitochondrial dysfunction and psychiatric-relevant behavioral deficits in spinocerebellar ataxia 1 mouse model

Spinocerebellar ataxia 1 (SCA1) is a devastating neurodegenerative disease associated with cerebellar degeneration and motor deficits. However, many patients also exhibit neuropsychiatric impairments such as depression and apathy; nevertheless, the existence of a causal link between the psychiatric symptoms and SCA1 neuropathology remains controversial. This study aimed to explore behavioral deficits in a knock-in mouse SCA1 (SCA1154Q/2Q) model and to identify the underlying neuropathology. We found that the SCA1 mice exhibit previously undescribed behavioral impairments such as increased anxiety- and depressive-like behavior and reduced prepulse inhibition and cognitive flexibility. Surprisingly, non-motor deficits characterize the early SCA1 stage in mice better than does ataxia. Moreover, the SCA1 mice exhibit significant hippocampal atrophy with decreased plasticity-related markers and markedly impaired neurogenesis. Interestingly, the hippocampal atrophy commences earlier than the cerebellar degeneration and directly reflects the individual severity of some of the behavioral deficits. Finally, mitochondrial respirometry suggests profound mitochondrial dysfunction in the hippocampus, but not in the cerebellum of the young SCA1 mice. These findings imply the essential role of hippocampal impairments, associated with profound mitochondrial dysfunction, in SCA1 behavioral deficits. Moreover, they underline the view of SCA1 as a complex neurodegenerative disease and suggest new avenues in the search for novel SCA1 therapies.

Nitric Oxide Signaling Strengthens Inhibitory Synapses of Cerebellar Molecular Layer Interneurons through a GABARAP-Dependent Mechanism.

Nitric oxide (NO) is an important signaling molecule that fulfills diverse functional roles as a neurotransmitter or diffusible second messenger in the developing and adult CNS. Although the impact of NO on different behaviors such as movement, sleep, learning, and memory has been well documented, the identity of its molecular and cellular targets is still an area of ongoing investigation. Here, we identify a novel role for NO in strengthening inhibitory GABAA receptor-mediated transmission in molecular layer interneurons of the mouse cerebellum. NO levels are elevated by the activity of neuronal NO synthase (nNOS) following Ca2+ entry through extrasynaptic NMDA-type ionotropic glutamate receptors (NMDARs). NO activates protein kinase G with the subsequent production of cGMP, which prompts the stimulation of NADPH oxidase and protein kinase C (PKC). The activation of PKC promotes the selective strengthening of α3-containing GABAARs synapses through a GΑΒΑ receptor-associated protein-dependent mechanism. Given the widespread but cell type-specific expression of the NMDAR/nNOS complex in the mammalian brain, our data suggest that NMDARs may uniquely strengthen inhibitory GABAergic transmission in these cells through a novel NO-mediated pathway.SIGNIFICANCE STATEMENT Long-term changes in the efficacy of GABAergic transmission is mediated by multiple presynaptic and postsynaptic mechanisms. A prominent pathway involves crosstalk between excitatory and inhibitory synapses whereby Ca2+-entering through postsynaptic NMDARs promotes the recruitment and strengthening of GABAA receptor synapses via Ca2+/calmodulin-dependent protein kinase II. Although Ca2+ transport by NMDARs is also tightly coupled to nNOS activity and NO production, it has yet to be determined whether this pathway affects inhibitory synapses. Here, we show that activation of NMDARs trigger a NO-dependent pathway that strengthens inhibitory GABAergic synapses of cerebellar molecular layer interneurons. Given the widespread expression of NMDARs and nNOS in the mammalian brain, we speculate that NO control of GABAergic synapse efficacy may be more widespread than has been appreciated.

Evaluation of Phytosomal Curcumin as an Anti-inflammatory Agent for Chronic Glial Activation in the GFAP-IL6 Mouse Model.

Chronic glial activation is characterized by an increased number of activated microglia and astroglia; these secrete free radicals and cytotoxic cytokines, subsequently causing neuronal damage. This study investigated the hypothesis that a soy-lecithin based phytosomal curcumin formulation can decrease glial activation in the brains of GFAP-IL6 mice, a model of chronic glial activation, which exhibits gliosis in various regions of the brain. Three doses of Meriva curcumin (MC) (874, 436, and 218 PPM) were fed to 3-month-old GFAP-IL6 and wild-type (WT) mice for 4 weeks. As markers of glial activation, the total numbers of Iba-1+ and TSPO+ microglia and macrophages, and GFAP+ astrocytes, were determined in the cerebellum and hippocampus by immunohistochemistry and unbiased stereology. Furthermore, the morphology of the glial cells was assessed by confocal microscopy and Sholl analysis. Administration of phytosomal curcumin led to a dose-dependent reduction in neuroinflammatory markers. Phytosomal curcumin (874 PPM) decreased the number of microglia by 26.2% in the hippocampus and by 48% in the cerebellum of the GFAP-IL6 mice compared with the GFAP-IL6 mice on normal food. Additionally, GFAP+ astrocyte numbers in the hippocampus of the GFAP-IL6 mice were decreased by 42%. The GFAP-IL6 mice exhibited a different microglial morphology to the WT mice, showing an increased soma size and perimeter. This difference was significantly reduced by the 874 PPM phytosomal curcumin dose. Our findings demonstrate that phytosomal curcumin is able to attenuate the inflammatory pathology, and potentially reverse the detrimental effects of chronic glial activation.

Components of Endocannabinoid Signaling System Are Expressed in the Perinatal Mouse Cerebellum and Required for Its Normal Development.

Endocannabinoid (eCB) signaling system (ECS), encompassing cannabinoid receptors and enzymes involved in the synthesis and degradation of the endogenous cannabinoid signaling lipids, is highly expressed in the cerebellar cortex of adult humans and rodents. In addition to their well-established role in neuromodulation, eCBs have been shown to play key roles in aspects of neurodevelopment in the fore- and mid-brain, including neurogenesis, cell migration, and synapse specification. However, little is known about the role of ECS in cerebellar development. In this study, we conducted immunohistochemical characterization of ECS components through key stages of cerebellar development in mice using antibodies for 2-arachidonoylglycerol (2-AG) synthetizing and degrading enzymes and the major brain cannabinoid receptor, cannabinoid receptor 1 (CB1), in combination with cerebellar cell markers. Our results reveal a temporally, spatially, and cytologically dynamic pattern of expression. Production, receptor binding, and degradation of eCBs are tightly controlled, thus localization of eCB receptors and the complementary cannabinoid signaling machinery determines the direction, duration, and ultimately the outcome of eCB signaling. To gain insights into the role of eCB signaling in cerebellar development, we characterized gross anatomy of cerebellar midvermis in CB1 knock-out (CB1 KO) mice, as well as their performance in cerebellar-influenced motor tasks. Our results show persistent and selective anatomic and behavioral alterations in CB1 KOs. Consequently, the insights gained from this study lay down the foundation for investigating specific cellular and molecular mechanisms regulated by eCB signaling during cerebellar development.

Acute ethanol exposure rapidly alters cerebellar and cortical microglial physiology.

Alcohol use is highly prevalent in modern society and ramifications of alcohol abuse pose a large public health concern. Previous work investigating the effects of alcohol exposure on the brain has implicated microglia, the resident immune cells of the central nervous system (CNS), as critical participants in the brain's response to chronic and developmental ethanol (EtOH) exposure. As rapid sensors of their environment, microglia also have the capacity to rapidly respond to alcohol administration and to contribute to acute effects of alcohol on the brain; however, their acute responses have not been assessed. Here, for the first time, we have examined the acute response of microglia to alcohol intoxication in vivo utilizing two-photon microscopy to assess the dynamics of these motile cells in both visual cortex and the cerebellum of mice. We found that microglia respond rapidly to EtOH exposure with fast changes in morphology, motility, parenchyma surveillance, and injury response. However, regional differences between the responses of cerebellar and cortical microglial populations indicate that subtle differences in microglial physiology may alter their vulnerability to acute alcohol intoxication. Our findings suggest that the longer-term effects of repeated EtOH exposure on microglia may result from repeat acute alterations in microglial physiology by single exposure to alcohol which rapidly alter behavior in specific microglial populations.

Cerebellum-enriched protein INPP5A contributes to selective neuropathology in mouse model of spinocerebellar ataxias type 17

Spinocerebellar ataxias 17 (SCA17) is caused by polyglutamine (polyQ) expansion in the TATA box-binding protein (TBP). The selective neurodegeneration in the cerebellum in SCA17 raises the question of why ubiquitously expressed polyQ proteins can cause neurodegeneration in distinct brain regions in different polyQ diseases. By expressing mutant TBP in different brain regions in adult wild-type mice via stereotaxic injection of adeno-associated virus, we found that adult cerebellar neurons are particularly vulnerable to mutant TBP. In SCA17 knock-in mice, mutant TBP inhibits SP1-mediated gene transcription to down-regulate INPP5A, a protein that is highly abundant in the cerebellum. CRISPR/Cas9-mediated deletion of Inpp5a in the cerebellum of wild-type mice leads to Purkinje cell degeneration, and Inpp5a overexpression decreases inositol 1,4,5-trisphosphate (IP3) levels and ameliorates Purkinje cell degeneration in SCA17 knock-in mice. Our findings demonstrate the important contribution of a tissue-specific protein to the polyQ protein-mediated selective neuropathology.

Abnormalities in the cerebellar levels of trophic factors BDNF and GDNF in pcd and lurcher cerebellar mutant mice

Hereditary cerebellar degenerations are severe and complex diseases for which there is currently no effective causal treatment. A hopeful method could be the support of plasticity or neurotransplantation. However, there are still many unknown aspects which could influence the outcome of treatment. As neurotrophic factors are essential in neuroplasticity and neuronal integration, potential abnormalities in their levels could be involved in the pathogenesis of the disease and would possibly explain the unsuitability of diseased cerebellum for the graft integration. The aim of this study was to identify and compare basal levels of trophic factors BDNF and GDNF in the cerebellum in two mouse models of cerebellar degeneration – Lurcher and pcd. Basal levels of BDNF in the cerebellum have been shown to be lower in both mutant models than in healthy controls. However, the GDNF levels were surprisingly increased in the cerebella of Lurcher mutant mice compared to both wild type and pcd mice. In addition, a different distribution of GFAP-positive cells in the cerebellum was revealed in Lurcher mice. These differences suggest that the niche of the Lurcher mutant cerebellum is changed. The question, however, remains how these changes are related to the neurodegenerative process and how they could influence potential compensatory mechanisms, plasticity and response to therapeutic interventions.

Effects of a solid lipid curcumin particle formulation on chronic activation of microglia and astroglia in the GFAP-IL6 mouse model

Chronic glial activation is characterized by increased numbers of activated glial cells, secreting free radicals and cytotoxic cytokines, subsequently causing neuronal damage. In order to investigate the anti-inflammatory activity of Longvida® Optimised Curcumin (LC), we fed 500 ppm of LC to 2-month-old wild type and GFAP-IL6 mice for 6 months. LC feeding led to a significant reduction in the number of Iba-1+ microglia by 26% in the hippocampus and by 48% in the cerebellum, GFAP+ astrocytes by 30%, and TSPO+ cells by 24% in the hippocampus and by 31% in the cerebellum of the GFAP-IL6 mice. The morphology of the cells was assessed and LC significantly decreased the dendritic length of microglia and the convex area, convex perimeter, dendritic length, nodes and number of processes of astrocytes in the hippocampus while decreasing the soma area and perimeter in the cerebellum, in LC-fed GFAP-IL6 mice. In addition, LC feeding increased pre- and postsynaptic protein levels and improved balance measured by Rotarod. Together, these data suggest that LC is able to attenuate the inflammatory pathology and ameliorate neurodegeneration and motor deficits in GFAP-IL6 mice. For patients with neuro-inflammatory disorders, LC might potentially reverse the detrimental effects of chronic glial activation.

DGKγ Knock-Out Mice Show Impairments in Cerebellar Motor Coordination, LTD, and the Dendritic Development of Purkinje Cells through the Activation of PKCγ.

Diacylglycerol kinase γ (DGKγ) regulates protein kinase C (PKC) activity by converting DG to phosphatidic acid (PA). DGKγ directly interacts with PKCγ and is phosphorylated by PKCγ, resulting in the upregulation of lipid kinase activity. PKC dysfunction impairs motor coordination, indicating that the regulation of PKC activity is important for motor coordination. DGKγ and PKC are abundantly expressed in cerebellar Purkinje cells. However, the physiological role of DGKγ has not been elucidated. Therefore, we developed DGKγ knock-out (KO) mice and tested their cerebellar motor coordination. In DGKγ KO mice, cerebellar motor coordination and long-term depression (LTD) were impaired, and the dendrites of Purkinje cells from DGKγ KO mice were significantly retracted. Interestingly, treatment with the cPKC inhibitor Gö6976 (Gö) rescued the dendritic retraction of primary cultured Purkinje cells from DGKγ KO mice. In contrast, treatment with the PKC activator 12-o-tetradecanoylphorbol 13-acetate (TPA) reduced morphologic alterations in the dendrites of Purkinje cells from wild-type (WT) mice. In addition, we confirmed the upregulation of PKCγ activity in the cerebellum of DGKγ KO mice and rescued impaired LTD in DGKγ KO mice with a PKCγ-specific inhibitor. Furthermore, impairment of motor coordination observed in DGKγ KO mice was rescued in tm1c mice with DGKγ reexpression induced by the FLP-flippase recognition target (FRT) recombination system. These results indicate that DGKγ is involved in cerebellar LTD and the dendritic development of Purkinje cells through the regulation of PKCγ activity, and thus contributes to cerebellar motor coordination.