Memantine suppresses the excitotoxicity but fails to rescue the ataxic phenotype in SCA1 model mice.

Spinocerebellar ataxia type 1 (SCA1) is a debilitating neurodegenerative disorder of the cerebellum and brainstem. Memantine has been proposed as a potential treatment for SCA1. It blocks N-methyl-D-aspartate (NMDA) receptors on neurons, reduces excitotoxicity and decreases neurodegeneration in Alzheimer models. However, in cerebellar neurodegenerative diseases, the potential value of memantine is still unclear. We investigated the effects of memantine on motor performance and synaptic transmission in the cerebellum in a mouse model where mutant ataxin 1 is specifically targeted to glia. Lentiviral vectors (LVV) were used to express mutant ataxin 1 selectively in Bergmann glia (BG). In mice transduced with the mutant ataxin 1, chronic treatment with memantine improved motor activity during initial tests, presumably due to preserved BG and Purkinje cell (PC) morphology and numbers. However, mice were unable to improve their rota rod scores during next days of training. Memantine also compromised improvement in the rota rod scores in control mice upon repetitive training. These effects may be due to the effects of memantine on plasticity (LTD suppression) and NMDA receptor modulation. Some effects of chronically administered memantine persisted even after its wash-out from brain slices. Chronic memantine reduced morphological signs of neurodegeneration in the cerebellum of SCA1 model mice. This resulted in an apparent initial reduction of ataxic phenotype, but memantine also affected cerebellar plasticity and ultimately compromised motor learning. We speculate that that clinical application of memantine in SCA1 might be hampered by its ability to suppress NMDA-dependent plasticity in cerebellar cortex.

Cerebellar and Olfactory Bulb Perturbations Induced by Vanadium Neurotoxicity in the African Giant Rat (Cricetomys gambianus, Waterhouse).

The African giant rat, AGR (Cricetomys gambianus) is a unique rodent known for its keen sense of smell which has enabled its use in the diagnosis of tuberculosis and demining activities in war torn countries. This keen sense of smell and the ability to navigate tight spaces are skills modulated by the olfactory bulb and cerebellum. While the brain is generally susceptible to environmental pollutants such as heavy metals, vanadium has predilection for these two brain regions. This work was thus designed to investigate the probable neurotoxic effect of vanadium on the neuronal cytoarchitecture of the cerebellum and olfactory bulb in this rodent. To achieve this, twelve adults male AGRs were divided into two groups (vanadium and control groups) and were given intraperitoneal injections of 3mg/kg body weight sodium metavanadate and normal saline respectively for 14 days. After which they were sacrificed, and brains harvested for histological investigations using Nissl and Golgi staining techniques. Results from our experiment revealed Purkinje cell degeneration and pyknosis as revealed by a lower intact-pyknotic cell (I-P) ratio, higher pyknotic Purkinje cell density and poor dendritic arborizations in the molecular layer of the cerebellum in the vanadium treated group. In the olfactory bulb, neuronal loss in the glomerular layer was observed as shrunken glomeruli. These neuronal changes have been linked to deficits in motor function and disruption of odor transduction in the olfactory bulb. This work has further demonstrated the neurotoxic effects of vanadium on the cerebellum and olfactory bulb of the AGR and the likely threat it may pose to the translational potentials of this rodent. We therefore propose the use of this rodent as a suitable model for better understanding vanadium induced olfactory and cerebellar dysfunctions.

Modified climbing fiber/Purkinje cell synaptic connectivity in the cerebellum of the neonatal phencyclidine model of schizophrenia.

Environmental perturbations during the first years of life are a major factor in psychiatric diseases. Phencyclidine (PCP), a drug of abuse, has psychomimetic effects, and neonatal subchronic administration of PCP in rodents leads to long-term behavioral changes relevant for schizophrenia. The cerebellum is increasingly recognized for its role in diverse cognitive functions. However, little is known about potential cerebellar changes in models of schizophrenia. Here, we analyzed the characteristics of the cerebellum in the neonatal subchronic PCP model. We found that, while the global cerebellar cytoarchitecture and Purkinje cell spontaneous spiking properties are unchanged, climbing fiber/Purkinje cell synaptic connectivity is increased in juvenile mice. Neonatal subchronic administration of PCP is accompanied by increased cFos expression, a marker of neuronal activity, and transient modification of the neuronal surfaceome in the cerebellum. The largest change observed is the overexpression of Ctgf, a gene previously suggested as a biomarker for schizophrenia. This neonatal increase in Ctgf can be reproduced by increasing neuronal activity in the cerebellum during the second postnatal week using chemogenetics. However, it does not lead to increased climbing fiber/Purkinje cell connectivity in juvenile mice, showing the complexity of PCP action. Overall, our study shows that administration of the drug of abuse PCP during the developmental period of intense cerebellar synaptogenesis and circuit remodeling has long-term and specific effects on Purkinje cell connectivity and warrants the search for this type of synaptic changes in psychiatric diseases.

Stereological Study on the Effect of Carnosine on of Purkinje Cells in the Cerebellum of Rats Exposed to 900 MHz Electromagnetic Field.

AIM: To evaluate the impact of carnosine on Purkinje neurons in rats exposed to a 900 Mhz electromagnetic field. MATERIAL AND METHODS: This study evaluated 24 rats divided into the following three different groups: a control group, a group exposed to the electromagnetic field, and a group that was injected with carnosine while being exposed to the electromagnetic field. The electromagnetic field group was exposed to a 900 Mhz electromagnetic field for an hour daily over 28 days. Thereafter, stereological analysis was performed histologically on cerebellar sections, and the number of Purkinje cells were counted. RESULTS: The electromagnetic field group had remarkably fewer Purkinje cell compared to control. The electromagnetic field group plus 20 mg of carnosine had significantly more total Purkinje cells compared to the electromagnetic field group (p < 0.05). CONCLUSION: The present study showed that electromagnetic field exposure decreases the number of Purkinje cell, whereas carnosine protected the cerebellum from neural damage induced by electromagnetic field exposure.

Anti-Excitotoxic Effects of N-Butylidenephthalide Revealed by Chemically Insulted Purkinje Progenitor Cells Derived from SCA3 iPSCs.

Spinocerebellar ataxia type 3 (SCA3) is characterized by the over-repetitive CAG codon in the ataxin-3 gene (ATXN3), which encodes the mutant ATXN3 protein. The pathological defects of SCA3 such as the impaired aggresomes, autophagy, and the proteasome have been reported previously. To date, no effective treatment is available for SCA3 disease. This study aimed to study anti-excitotoxic effects of n-butylidenephthalide by chemically insulted Purkinje progenitor cells derived from SCA3 iPSCs. We successfully generated Purkinje progenitor cells (PPs) from SCA3 patient-derived iPSCs. The PPs, expressing both neural and Purkinje progenitor's markers, were acquired after 35 days of differentiation. In comparison with the PPs derived from control iPSCs, SCA3 iPSCs-derived PPs were more sensitive to the excitotoxicity induced by quinolinic acid (QA). The observations of QA-treated SCA3 PPs showing neural degeneration including neurite shrinkage and cell number decrease could be used to quickly and efficiently identify drug candidates. Given that the QA-induced neural cell death of SCA3 PPs was established, the activity of calpain in SCA3 PPs was revealed. Furthermore, the expression of cleaved poly (ADP-ribose) polymerase 1 (PARP1), a marker of apoptotic pathway, and the accumulation of ATXN3 proteolytic fragments were observed. When SCA3 PPs were treated with n-butylidenephthalide (n-BP), upregulated expression of calpain 2 and concurrent decreased level of calpastatin could be reversed, and the overall calpain activity was accordingly suppressed. Such findings reveal that n-BP could not only inhibit the cleavage of ATXN3 but also protect the QA-induced excitotoxicity from the Purkinje progenitor loss.

CD8 T-cell-mediated cerebellitis directed against Purkinje cell antigen after ipilimumab for small cell lung cancer.

We report a rapidly progressive and fatal CD8 T-cell-mediated cerebellitis after ipilimumab (cytotoxic T-lymphocyte-associated protein 4 inhibitor) for small cell lung cancer. Clinical features and histopathology were consistent with an accelerated form of paraneoplastic cerebellar degeneration. A patchy CD8 T-cell infiltrate spatially corresponded to areas of Purkinje cell loss, with occasional CD8 polarisation towards Purkinje cells. CD20-positive B cells were sparse. CD8 T-cell-mediated cerebellitis after immune checkpoint inhibitor treatment may recapitulate the early stages of paraneoplastic cerebellar degeneration.

Structure-Activity Relationship Study of Subtype-Selective Positive Modulators of KCa2 Channels.

A series of modified N-cyclohexyl-2-(3,5-dimethyl-1H-pyrazol-1-yl)-6-methylpyrimidin-4-amine (CyPPA) analogues were synthesized by replacing the cyclohexane moiety with different 4-substituted cyclohexane rings, tyrosine analogues, or mono- and dihalophenyl rings and were subsequently studied for their potentiation of KCa2 channel activity. Among the N-benzene-N-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-4-pyrimidinamine derivatives, halogen decoration at positions 2 and 5 of benzene-substituted 4-pyrimidineamine in compound 2q conferred a ∼10-fold higher potency, while halogen substitution at positions 3 and 4 of benzene-substituted 4-pyrimidineamine in compound 2o conferred a ∼7-fold higher potency on potentiating KCa2.2a channels, compared to that of the parent template CyPPA. Both compounds retained the KCa2.2a/KCa2.3 subtype selectivity. Based on the initial evaluation, compounds 2o and 2q were selected for testing in an electrophysiological model of spinocerebellar ataxia type 2 (SCA2). Both compounds were able to normalize the abnormal firing of Purkinje cells in cerebellar slices from SCA2 mice, suggesting the potential therapeutic usefulness of these compounds for treating symptoms of ataxia.

Opioid receptors modulate parallel fiber-Purkinje cell synaptic transmission in mouse cerebellum.

Opioid receptors play important roles in, among others, learning and memory, emotional responses, addiction, and pain. In recent years, the cerebellum has received increasing attention for its role in non-motor functions. The Purkinje cell (PC) is the only efferent neuron in the cerebellar cortex, and receives glutamatergic synaptic inputs from the parallel fibers (PF) formed by the axons of granule cells. Studies have shown that opioid receptors are expressed during the development of cerebellar cells. However, the distribution of opioid receptors, their subtypes in cerebellar PF-PC synapses, and their effects on synaptic transmission remain unclear. To examine these questions, we used whole-cell patch clamp recordings and pharmacological methods to determine the effects of activating three different opioid receptor subtypes on synaptic transmission at PF-PC synapses. In the presence of picrotoxin, mouse cerebellar slices were perfused with agonists or blockers of different opioid receptor subtypes, and the changes in excitatory postsynaptic currents (EPSCs) were examined. Both agonists of µ-opioid receptors (MOR) and δ-opioid receptors (DOR) significantly reduced the amplitude and area under the curve of PF-PC EPSCs in a concentration-dependent manner, accompanied by an increase in the paired-pulsed ratio (PPR). These effects could be blocked by respective receptor antagonists. In contrast, no significant changes were found after the application of κ-opioid receptor (KOR) agonists. In conclusion, MOR and DOR are present at the axon terminals of PF in the mouse cerebellar cortex, whereas no or negligible amounts of KOR are found. Activation of MOR and DOR regulates PF-PC synaptic transmission via inhibition of glutamate (Glu) release in cerebellar cortex in mice. We also found that endogenous opioid peptides are present in PF-PC synapses of mouse cerebellum, which also can inhibit the release of Glu.

Chronic ethanol exposure during adolescence impairs simple spike activity of cerebellar Purkinje cells in vivo in mice.

Cerebellar Purkinje cells (PCs) play critical roles in motor coordination and motor learning through their simple spike (SS) activity. Previous studies have shown that chronic ethanol exposure (CEE) in adolescents impairs learning, attention, and behavior, at least in part by impairing the activity of cerebellar PCs. In this study, we investigated the effect of CEE on the SS activity in urethane-anesthetized adolescent mice by in vivo electrophysiological recordings and pharmacological methods. Our results showed that the cerebellar PCs in CEE adolescent mice expressed a significant decrease in the frequency and an increase in the coefficient of variation (CV) of SS than control group. Blockade of ɤ-aminobutyric acid A (GABAA) receptor did not change the frequency and CV of SS firing in control group but produced a significant increase in the frequency and a decrease in the CV of SS firing in CEE mice. The CEE-induced decrease in SS firing rate and increase in CV were abolished by application of an N-methyl-D-aspartate (NMDA) receptor blocker, D-APV, but not by anα-amino-3-hydroxy-5-methyl -4-isoxazolepropionic acid (AMPA) receptor antagonist, NBQX. Notably, the spontaneous spike rate of molecular layer interneurons (MLIs) in CEE mice was significantly higher than control group, which was also abolished by application of D-APV. These results indicate that adolescent CEE enhances the spontaneous spike firing rate of MLIs through activation of NMDA receptor, resulting in a depression in the SS activity of cerebellar PCs in vivo in mice.

Cholecystokinin 1 receptor activation restores normal mTORC1 signaling and is protective to Purkinje cells of SCA mice.

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.

Expression Pattern of T-Type Ca2+ Channels in Cerebellar Purkinje Cells after VEGF Treatment.

T-type Ca2+ channels, generating low threshold calcium influx in neurons, play a crucial role in the function of neuronal networks and their plasticity. To further investigate their role in the complex field of research in plasticity of neurons on a molecular level, this study aimed to analyse the impact of the vascular endothelial growth factor (VEGF) on these channels. VEGF, known as a player in vasculogenesis, also shows potent influence in the central nervous system, where it elicits neuronal growth. To investigate the influence of VEGF on the three T-type Ca2+ channel isoforms, Cav3.1 (encoded by Cacna1g), Cav3.2 (encoded by Cacna1h), and Cav3.3 (encoded by Cacna1i), lasermicrodissection of in vivo-grown Purkinje cells (PCs) was performed, gene expression was analysed via qPCR and compared to in vitro-grown PCs. We investigated the VEGF receptor composition of in vivo- and in vitro-grown PCs and underlined the importance of VEGF receptor 2 for PCs. Furthermore, we performed immunostaining of T-type Ca2+ channels with in vivo- and in vitro-grown PCs and showed the distribution of T-type Ca2+ channel expression during PC development. Overall, our findings provide the first evidence that the mRNA expression of Cav3.1, Cav3.2, and Cav3.3 increases due to VEGF stimulation, which indicates an impact of VEGF on neuronal plasticity.

Protective Effect of Memantine on Bergmann Glia and Purkinje Cells Morphology in Optogenetic Model of Neurodegeneration in Mice.

Spinocerebellar ataxias are a family of fatal inherited diseases affecting the brain. Although specific mutated proteins are different, they may have a common pathogenetic mechanism, such as insufficient glutamate clearance. This function fails in reactive glia, leading to excitotoxicity and overactivation of NMDA receptors. Therefore, NMDA receptor blockers could be considered for the management of excitotoxicity. One such drug, memantine, currently used for the treatment of Alzheimer's disease, could potentially be used for the treatment of other forms of neurodegeneration, for example, spinocerebellar ataxias (SCA). We previously demonstrated close parallels between optogenetically induced cerebellar degeneration and SCA1. Here we induced reactive transformation of cerebellar Bergmann glia (BG) using this novel optogenetic approach and tested whether memantine could counteract changes in BG and Purkinje cell (PC) morphology and expression of the main glial glutamate transporter-excitatory amino acid transporter 1 (EAAT1). Reactive BG induced by chronic optogenetic stimulation presented increased GFAP immunoreactivity, increased thickness and decreased length of its processes. Oral memantine (~90 mg/kg/day for 4 days) prevented thickening of the processes (1.57 to 1.81 vs. 1.62 µm) and strongly antagonized light-induced reduction in their average length (186.0 to 150.8 vs. 171.9 µm). Memantine also prevented the loss of the key glial glutamate transporter EAAT1 on BG. Finally, memantine reduced the loss of PC (4.2 ± 0.2 to 3.2 ± 0.2 vs. 4.1 ± 0.3 cells per 100 µm of the PC layer). These results identify memantine as potential neuroprotective therapeutics for cerebellar ataxias.

Metanil yellow promotes oxidative stress, astrogliosis, and apoptosis in the cerebellar cortex of adult male rat with possible protective effect of scutellarin: A histological and immunohistochemical study.

Metanil yellow is a food dye that has harmful impacts on different body systems. Scutellarin has antioxidant, antiapoptotic, and anti-inflammatory activities. The aim of the current research was to study the effect of chronic administration of metanil yellow on the cerebellar cortex of rats and to evaluate the protective effect of scutellarin. Forty adult male rats were allocated into four groups: group I acted as control, group II was administrated scutellarin (100 mg/kg/day), group III was administrated metanil yellow (200 mg/kg/day), and group IV was administrated scutellarin and metanil yellow as in group II and group III. The agents were administered via oral gavage for 8 weeks. Metanil yellow induced a significant rise in the malondialdehyde coupled with a significant reduction in the superoxide dismutase and glutathione peroxidase. The Purkinje cells were irregular and shrunken with condensed nuclei. A significant elevation in glial fibrillary acidic protein (GFAP) and cleaved caspase-3 as well as a significant reduction of synaptophysin expression were revealed in comparison with the control group. Interestingly, few changes were noticed in rats given metanil yellow concomitant with scutellarin. In conclusion, scutellarin could protect against metanil yellow-induced alterations in the cerebellar cortex by reducing oxidative stress and minimizing gliosis.

n-Butylidenephthalide Modulates Autophagy to Ameliorate Neuropathological Progress of Spinocerebellar Ataxia Type 3 through mTOR Pathway.

Spinocerebellar ataxia type 3 (SCA3), a hereditary and lethal neurodegenerative disease, is attributed to the abnormal accumulation of undegradable polyglutamine (polyQ), which is encoded by mutated ataxin-3 gene (ATXN3). The toxic fragments processed from mutant ATXN3 can induce neuronal death, leading to the muscular incoordination of the human body. Some treatment strategies of SCA3 are preferentially focused on depleting the abnormal aggregates, which led to the discovery of small molecule n-butylidenephthalide (n-BP). n-BP-promoted autophagy protected the loss of Purkinje cell in the cerebellum that regulates the network associated with motor functions. We report that the n-BP treatment may be effective in treating SCA3 disease. n-BP treatment led to the depletion of mutant ATXN3 with the expanded polyQ chain and the toxic fragments resulting in increased metabolic activity and alleviated atrophy of SCA3 murine cerebellum. Furthermore, n-BP treated animal and HEK-293GFP-ATXN3-84Q cell models could consistently show the depletion of aggregates through mTOR inhibition. With its unique mechanism, the two autophagic inhibitors Bafilomycin A1 and wortmannin could halt the n-BP-induced elimination of aggregates. Collectively, n-BP shows promising results for the treatment of SCA3.

The protective effect of nnano-selenium against cadmium-induced cerebellar injury via the heat shock protein pathway in chicken.

Cadmium (Cd) is one of the toxic environmental heavy metals that poses health hazard to animals due to its toxicity. Nano-Selenium (Nano-Se) is a Nano-composite form of Se, which has emerged as a promising therapeutic agent for its protective roles against heavy metals-induced toxicity. Heat shock proteins (HSPs) play a critical role in cellular homeostasis. However, the potential protective effects of Nano-Se against Cd-induced cerebellar toxicity remain to be illustrated. To investigate the toxic effects of Cd on chicken's cerebellum, and the protective effects of Nano-Se against Cd-induced cerebellar toxicity, a total of 80 male chicks were divided into four groups and treated as follows: (A) 0 mg/kg Cd, (B) 1 mg/kg Nano-Se (C) 140 mg/kg Cd + 1 mg/kg Nano-Se (D) 140 mg/kg Cd for 90 days. We tested heat shock protein pathway-related factors including heat shock factors (HSFs) HSF1, HSF2, HSF3 and heat shock proteins (HSPs) HSP10, HSP25, HSP27, HSP40, HSP60, HSP70 and HSP90 expressions. Histopathological results showed that Cd treatment caused degradation of Purkinje cells. In addition, HSFs and HSPs expression decreased significantly in the Cd group. Nano-Se co-treatment with Cd enhanced the expression of HSFs and HSPs. In summary, our findings explicated a potential protective effect of Nano-Se against Cd-induced cerebellar injury in chicken, suggesting that Nano-Se is a promising therapeutic agent for the treatment of Cd toxicity.

[Electrophysiological Effects of Ionophore-induced Increases in Intracellular Na+ in Cardiomyocytes].

Na ionophores increase intracellular Na+ ([Na+]i). Membrane potentials and currents were measured using microelectrode and whole-cell patch-clamp techniques. Monensin (10-6-3×10-5 M) reduced the slope of the pacemaker potentials and shortened the action potential duration (APD) in sino-atrial nodal and Purkinje cells. Monensin (10-5 M) shortened the APD and reduced the amplitude of the plateau phase in ventricular myocytes. Monensin decreased the hyperpolarization-activated inward current (If), and it increased the transient outward potassium current (Ito) in Purkinje cells. In addition, monensin decreased the sodium current (INa), shifting the inactivation curve to the hyperpolarized direction. Moreover, monensin decreased the L-type calcium current (ICa) in ventricular myocytes. The Na+-Ca2+ exchange current (INa-Ca) was augmented particularly in the reverse mode, and the Na+-K+ pump current (INa-K) was also activated by monensin in cardiomyocytes. The ATP-activated potassium current (IK,ATP) could be induced by monensin. Notably, the inward rectifying K+ current (IK1), and the slow delayed outward K+ current (IKs) were not affected evidently by monensin. Collectively, alteration of [Na+]i can influence the activities of various ion channels and transporters. Thus, the significance of altered [Na+]i should be taken into consideration in the action of drugs affecting [Na+]i such as digitalis, Na+ channel blockers, and Na+ channel activating agents.

Withania coagulans extract attenuates oxidative stress-mediated apoptosis of cerebellar purkinje neurons after ischemia/reperfusion injury.

Cerebral ischemia/reperfusion (I/R) is known to increase reactive oxygen species (ROS) generation, consequences of oxidative stress (OS), and neuronal death in the susceptible brain areas including the cerebellum. Newly, remarkable attention has been paid to a natural diet with the capability to scavenge ROS. Withania coagulans root extract (WCE) is rich in components with antioxidants properties. Therefore, this study aimed to evaluate the effect of WCE on cerebellar Purkinje cells (PCs) against OS-mediated apoptosis after I/R injury. In this experimental study 64 male adult Wistar rats were randomly divided into 4 groups (n = 16) as follows: control, sham, I/R, and WCE 1000 + I/R. I/R animals were pretreated with daily administration of hydro-alcoholic WCE (1000 mg/kg) or distilled water as a vehicle for 30 days before I/R injury. After 72 h, the animals were sacrificed, the cerebellum tissue was removed and used for biochemical (CAT, SOD, GPx, and MDA levels) and histopathological (Nissl and TUNEL staining) assays. Findings showed that the MDA level and the number of apoptotic neurons significantly increased and viable Purkinje neurons decreased in I/R injury (p < 0.05). Administration of 1000 mg/kg WCE reduced MDA level and enhanced antioxidants activity including CAT, SOD, and GPx significantly. In addition, intact surviving PCs increased. At the same time, TUNEL-positive neurons decreased significantly in the WCE pre-treated group (p < 0.05). These findings suggest that WCE can counteract cerebral I/R-induced OS and associated neuronal death by enhancement of ROS scavenging and antioxidant capacity. It appears that pre-treatment with 1000 mg/kg WCE for thirty days can protect PCs against OS-mediated apoptosis after I/R injury.

Enrichment of NPC1-deficient cells with the lipid LBPA stimulates autophagy, improves lysosomal function, and reduces cholesterol storage.

Niemann-Pick C (NPC) is an autosomal recessive disorder characterized by mutations in the NPC1 or NPC2 genes encoding endolysosomal lipid transport proteins, leading to cholesterol accumulation and autophagy dysfunction. We have previously shown that enrichment of NPC1-deficient cells with the anionic lipid lysobisphosphatidic acid (LBPA; also called bis(monoacylglycerol)phosphate) via treatment with its precursor phosphatidylglycerol (PG) results in a dramatic decrease in cholesterol storage. However, the mechanisms underlying this reduction are unknown. In the present study, we showed using biochemical and imaging approaches in both NPC1-deficient cellular models and an NPC1 mouse model that PG incubation/LBPA enrichment significantly improved the compromised autophagic flux associated with NPC1 disease, providing a route for NPC1-independent endolysosomal cholesterol mobilization. PG/LBPA enrichment specifically enhanced the late stages of autophagy, and effects were mediated by activation of the lysosomal enzyme acid sphingomyelinase. PG incubation also led to robust and specific increases in LBPA species with polyunsaturated acyl chains, potentially increasing the propensity for membrane fusion events, which are critical for late-stage autophagy progression. Finally, we demonstrated that PG/LBPA treatment efficiently cleared cholesterol and toxic protein aggregates in Purkinje neurons of the NPC1I1061T mouse model. Collectively, these findings provide a mechanistic basis supporting cellular LBPA as a potential new target for therapeutic intervention in NPC disease.

Oleoylethanolamide Delays the Dysfunction and Death of Purkinje Cells and Ameliorates Behavioral Defects in a Mouse Model of Cerebellar Neurodegeneration.

Oleoylethanolamide (OEA) is an endocannabinoid that has been proposed to prevent neuronal damage and neuroinflammation. In this study, we evaluated the effects of OEA on the disruption of both cerebellar structure and physiology and on the behavior of Purkinje cell degeneration (PCD) mutant mice. These mice exhibit cerebellar degeneration, displaying microtubule alterations that trigger the selective loss of Purkinje cells and consequent behavioral impairments. The effects of different doses (1, 5, and 10 mg/kg, i.p.) and administration schedules (chronic and acute) of OEA were assessed at the behavioral, histological, cellular, and molecular levels to determine the most effective OEA treatment regimen. Our in vivo results demonstrated that OEA treatment prior to the onset of the preneurodegenerative phase prevented morphological alterations in Purkinje neurons (the somata and dendritic arbors) and decreased Purkinje cell death. This effect followed an inverted U-shaped time-response curve, with acute administration on postnatal day 12 (10 mg/kg, i.p.) being the most effective treatment regimen tested. Indeed, PCD mice that received this specific OEA treatment regimen showed improvements in motor, cognitive and social functions, which were impaired in these mice. Moreover, these in vivo neuroprotective effects of OEA were mediated by the PPARα receptor, as pretreatment with the PPARα antagonist GW6471 (2.5 mg/kg, i.p.) abolished them. Finally, our in vitro results suggested that the molecular effect of OEA was related to microtubule stability and structure since OEA administration normalized some alterations in microtubule features in PCD-like cells. These findings provide strong evidence supporting the use of OEA as a pharmacological agent to limit severe cerebellar neurodegenerative processes.

Disabling VEGF-Response of Purkinje Cells by Downregulation of KDR via miRNA-204-5p.

The vascular endothelial growth factor (VEGF) is well known for its wide-ranging functions, not only in the vascular system, but also in the central (CNS) and peripheral nervous system (PNS). To study the role of VEGF in neuronal protection, growth and maturation processes have recently attracted much interest. These effects are mainly mediated by VEGF receptor 2 (VEGFR-2). Current studies have shown the age-dependent expression of VEGFR-2 in Purkinje cells (PC), promoting dendritogenesis in neonatal, but not in mature stages. We hypothesize that microRNAs (miRNA/miR) might be involved in the regulation of VEGFR-2 expression during the development of PC. In preliminary studies, we performed a miRNA profiling and identified miR204-5p as a potential regulator of VEGFR-2 expression. In the recent study, organotypic slice cultures of rat cerebella (postnatal day (p) 1 and 9) were cultivated and VEGFR-2 expression in PC was verified via immunohistochemistry. Additionally, PC at age p9 and p30 were isolated from cryosections by laser microdissection (LMD) to analyse VEGFR-2 expression by quantitative RT-PCR. To investigate the influence of miR204-5p on VEGFR-2 levels in PC, synthetic constructs including short hairpin (sh)-miR204-5p cassettes (miRNA-mimics), were microinjected into PC. The effects were analysed by confocal laser scanning microscopy (CLSM) and morphometric analysis. For the first time, we could show that miR204-5p has a negative effect on VEGF sensitivity in juvenile PC, resulting in a significant decrease of dendritic growth compared to untreated juvenile PC. In mature PC, the overexpression of miR204-5p leads to a shrinkage of dendrites despite VEGF treatment. The results of this study illustrate, for the first time, which miR204-5p expression has the potential to play a key role in cerebellar development by inhibiting VEGFR-2 expression in PC.