Cerebellar Purkinje Research Articles

Dynamic molecular network analysis of iPSC-Purkinje cells differentiation delineates roles of ISG15 in SCA1 at the earliest stage

Better understanding of the earliest molecular pathologies of all neurodegenerative diseases is expected to improve human therapeutics. We investigated the earliest molecular pathology of spinocerebellar ataxia type 1 (SCA1), a rare familial neurodegenerative disease that primarily induces death and dysfunction of cerebellum Purkinje cells. Extensive prior studies have identified involvement of transcription or RNA-splicing factors in the molecular pathology of SCA1. However, the regulatory network of SCA1 pathology, especially central regulators of the earliest developmental stages and inflammatory events, remains incompletely understood. Here, we elucidated the earliest developmental pathology of SCA1 using originally developed dynamic molecular network analyses of sequentially acquired RNA-seq data during differentiation of SCA1 patient-derived induced pluripotent stem cells (iPSCs) to Purkinje cells. Dynamic molecular network analysis implicated histone genes and cytokine-relevant immune response genes at the earliest stages of development, and revealed relevance of ISG15 to the following degradation and accumulation of mutant ataxin-1 in Purkinje cells of SCA1 model mice and human patients.

Cerebellum Purkinje cell vulnerability in aged rats with memory impairment.

The cerebellum is involved in higher order cognitive function and is susceptible to age-related atrophy. However, limited evidence has directly examined the cerebellum's role in cognitive aging. To interrogate potential substrates of the relationship between cerebellar structure and memory in aging, here we target the Purkinje cells (PCs). The sole output neurons of the cerebellum, PC loss and/or degeneration underlie a variety of behavioral abnormalities. Using a rat model of normal cognitive aging, we immunostained sections through the cerebellum for the PC-specific protein, calbindin-D28k. Although morphometric quantification revealed no significant difference in total PC number as a function of age or cognitive status, regional cell number was a more robust correlate of memory performance in the young cerebellum than in aged animals. Parallel biochemical analysis of PC-specific protein levels in whole cerebellum additionally revealed that calbindin-D28k and Purkinje cell protein-2 (pcp-2) levels were lower selectively in aged rats with spatial memory impairment compared to both young animals and aged rats with intact memory. These results suggest that cognitive aging is associated with cerebellum vulnerability, potentially reflecting disruption of the cerebellum-medial temporal lobe network.

Chapter 8 - The episodic ataxias

The primary episodic ataxias (EAs) are a group of autosomal-dominant disorders characterized by transient recurrent incoordination and truncal instability, often triggered by physical exertion or emotional stress and variably associated with progressive baseline ataxia. There are now nine designated subtypes EA1-9 (OMIM) and late onset cerebellar ataxia with episodic features as newly designated SCA27B, based largely on genetic loci. Mutations have been identified in multiple individuals and families in 4 of the 9 EA subtypes, mostly with the onset before adulthood. This chapter focuses on the clinical assessment and management of EA, genetic diagnosis, and neurophysiologic consequences of the causative mutations in the best characterized EA syndromes: EA1 caused by mutations in KCNA1 encoding a neuronal voltage-gated potassium channel, EA2 caused by mutations in CACNA1A encoding a neuronal voltage-gated calcium channel, EA6 caused by mutations in SLC1A3 encoding a glutamate transporter that is also an anion channel, and SCA27B with late onset episodic ataxia caused by an intronic trinucleotide repeat in FGF14 encoding fibroblast growth factor 14 important in regulating the distribution of voltage-gated sodium channels in the cerebellar Purkinje and granule cells. The study of EA has illuminated previously unrecognized but important roles of ion channels and transporters in brain function with shared mechanisms underlying cerebellar ataxia, migraine, and epilepsy.

PERUBAHAN JUMLAH SEL PURKINJE SEREBELUM TIKUS WISTAR JANTAN SETELAH PEMAPARAN ROKOK ELEKTRIK

Design, taste, marketing and perceptions of safety from e-cigarettes have increased the appeal of e-cigarettes to young people around the world so that their use continues to increase. This is one of the threats in efforts to reduce the prevalence of smokers aged 10-18 years in Indonesia. Electric cigarettes cause oxidative stress while the brain is vulnerable to oxidative stress because it requires large amounts of oxygen. This experimental study with a posttest only control design aimed to determine changes in histological structure and number of male Wistar rat cerebellum Purkinje cells after exposure to electric cigarettes. This study used a sample of 32 male wistar rats (Rattus norvegicus) which were divided into 4 treatment groups: group A (without treatment), group B (put in an exposure cage), group C (given exposure to electric cigarettes 15 times in a cage exposure), and group D (given exposure to e-cigarettes 30 times in the exposure cage). Exposure to e-cigarettes was given for 50 days, then sacrificed and samples were made with Hematoxylin Eosyn (HE) staining. The results showed that the number of cerebellar purkinje cells in male Wistar rats exposed to electric cigarettes decreased significantly and there was a significant difference in the number of cells between 15 and 30 exposures and karyolysis occurred in the cells. E-cigarettes cause core damage and decrease in the number of Purkinje cells in the cerebellum of male wistar rats.

The enriched environment prevents degeneration of cerebellum Purkinje cells layer of rats.

Neurodegeneration is characterized by loss of neurons causing changes that lead individuals to debilitating conditions; the most common of this condition is the Alzheimer's disease. It has been related that enriched environment (EE) induces experience‑dependent plasticity mechanisms, improving the performance of the animals in learning and memory tests. This study evaluated the effects of EE on histological parameters of the cerebellum in rats that received intracerebroventricular streptozotocin. In the standard environment, streptozotocin (STZ) promoted a significant increase between the gaps in the Purkinje layer of approximately 20%. On the other hand, in an enriched environment, the control result (EE) was similar to the result under streptozotocin effect (STZEE). In the standard environment (SE) group a 26% significant reduction in Purkinje cell density was observed under STZ presence. By analyzing the results of the density of Purkinje cells under the effect of streptozotocin in a standard environment (STZSE) against the density of the layer of Purkinje cells also under the effect of streptozotocin in an enriched environment (STZEE), a significant reduction of approximately 76% in density was observed of Purkinje cells in standard environment (STZSE), the mean number of Purkinje cells in enriched environments was not reduced, despite of STZ. According to the results, treatment with STZ and exposure to EE did not change the cerebellum general morphology/cytoarchitecture, hence was no significant difference in the layers thickness. These facts demonstrate that the enriched environment appears to protect the Purkinje cells layer of cerebellum from possible degeneration.

1524-P: Selective Deletion of Nav1.7 in Cerebellar Purkinje Neurons Prevents Diet-Induced Obesity by Promoting Voluntary Locomotion

The pacemaker activity of cerebellum Purkinje neurons is well-known to be essential in multiple physiological functions, including voluntary locomotion, balance, aggressive behavior, and cognitive behavior. However, the specific ionic mechanism of Purkinje neurons in regulating voluntary locomotion is still unclear. To this end, we ex vivo electrophysiologically recorded the activity changes of Purkinje neurons after wheel running by using Pcp2-Cre/Rosa26-LTL-tdTOMATO reporter mice with tdTOMATO selectively expressed in Purkinje neurons. We found that the Purkinje neural activity was reduced by wheel running, associated with decreases in voltage-gated sodium current and expression of the voltage-gated sodium ion channel Nav1.7 (Scn9a), suggesting a potential role of Nav1.7-mediated sodium current. To test whether Nav1.7 is required for the activity dynamics of Purkinje neurons induced by voluntary wheel running and subsequent beneficial metabolic effects, we crossed Scn9a flox/flox mice with Pcp2-Cre mice to generate a mouse model with Nav1.7 selectively deleted from Purkinje neurons (Pcp2-Cre/Scn9a flox/flox, Nav1.7-KOPcp2). Nav1.7-KOPcp2 significantly decreased Purkinje neural dynamics, mimicking wheel running-induced neural activity reduction. Nav1.7-KOPcp2 mice also significantly increased voluntary locomotion compared to the control littermates. Further, Nav1.7-KOPcp2 mice showed decreased body weight gain, reduced blood glucose, and improved insulin sensitivity when fed a high-fat diet (HFD). These beneficial metabolic effects were associated with increased running activity but not food intake. In summary, these findings provide evidence to support the vital role of the Nav1.7 ion channel expressed by Purkinje neurons in the beneficial metabolic effects of voluntary locomotion activity. Disclosure B. Feng: None. P. Gao: None. J. Harms: None. P. Luo: None. P. Xu: None. Y. He: None. Funding National Institutes of Health (R01DK123098, R01DK129548)

Calsequestrin in Purkinje cells of mammalian cerebellum

Cerebellum is devoted to motor coordination and cognitive functions. Endoplasmic reticulum is the largest intracellular calcium store involved in all neuronal functions. Intralumenal calcium binding proteins play a pivotal role in calcium storage and contribute to both calcium release and uptake. Calsequestrin, a key calcium binding protein of sarco-endoplasmic reticulum in skeletal and cardiac muscles, was identified in chicken and fish cerebellum Purkinje cells, but its expression in mammals and human counterpart has not been studied in depth. Aim of the present paper was to investigate expression and localization of Calsequestrin in mammalian cerebellum. Calsequestrin was found to be expressed at low level in cerebellum, but specifically concentrated in Calbindin D28- and zebrin- immunopositive-Purkinje cells. Two additional fundamental calcium store markers, sarco-endoplasmic calcium pump isoform 2, SERCA2, and Inositol-trisphosphate receptor isoform 1, IP3R1, were found to be co-expressed in the region, with some localization peculiarities. In conclusion, a new marker was identified for Purkinje cells in adult mammals, including humans. Such a marker might help in staminal neuronal cells specification and in dissection of still unknown neurodegeneration and physio-pathological effects of dysregulated calcium homeostasis.

Correlation studies on pathological changes in brain with neurotransmitters and behavioural changes in Balb/c mice

Aim: To determine the plasma neurotransmitters simultaneously and to find any correlation with pathological changes in the hippocampus and Purkinje cells and their relation with behavioral changes in Balb/c mice. Methodology: In the present study, both sexes of Balb /C mice were divided into two groups (4 males and 4 females; n = 8): Both the groups were given a single dose of either saline or sodium valproate (400mg kg-1) respectively through subcutaneous injection on PND 14. Behavioural tests were conducted on mice pups on various postnatal days till 40th day. On PND 41, blood samples were collected from all the animals for quantification of the neurotransmitters (serotonin, dopamine, and noradrenalin) in plasma, animals were sacrificed by cervical dislocation and whole brain was isolated for histological examination of the Purkinje cells and hippocampus. Results: Sodium valproate exposed animals showed loss of motor skill development (delayed negative geotaxic response), increased locomotor activity, increased anxiety, and retardation in water maze performance, and lower social interaction. Histopathological evolutions of cerebellum purkinje cells and hippocampus showed 40-50% atrophic cells in sodium valproate animals compared to control animals. Interpretation: The results of the present study indicate that Sodium valproate changes specific brain cell population in Balb/C mice, which might be the reason for the altered neurotransmitter levels, leading to behavioural changes in these animals.

The AGTPBP1 gene in neurobiology.

The function of the Agtpbp1 gene has mainly been delineated by studying Agtpbp1pcd (pcd) mutant mice, characterized by losses in cerebellar Purkinje and granule cells along with degeneration of retinal photoreceptors, mitral cells of the olfactory bulb, thalamic neurons, and alpha-motoneurons. As a result of cerebellar degeneration, cerebellar GABA and glutamate concentrations in Agtpbp1pcd mutants decreased while monoamine concentrations increased. The salient behavioral phenotypes include cerebellar ataxia, a loss in motor coordination, and cognitive deficits. Similar neuropathogical and behavioral profiles have been described in childhood-onset human subjects with biallelic variants of AGTPBP1, including cerebellar ataxia and hypotonia.

Prediction of Neural Diameter From Morphology to Enable Accurate Simulation.

Accurate neuron morphologies are paramount for computational model simulations of realistic neural responses. Over the last decade, the online repository NeuroMorpho.Org has collected over 140,000 available neuron morphologies to understand brain function and promote interaction between experimental and computational research. Neuron morphologies describe spatial aspects of neural structure; however, many of the available morphologies do not contain accurate diameters that are essential for computational simulations of electrical activity. To best utilize available neuron morphologies, we present a set of equations that predict dendritic diameter from other morphological features. To derive the equations, we used a set of NeuroMorpho.org archives with realistic neuron diameters, representing hippocampal pyramidal, cerebellar Purkinje, and striatal spiny projection neurons. Each morphology is separated into initial, branching children, and continuing nodes. Our analysis reveals that the diameter of preceding nodes, Parent Diameter, is correlated to diameter of subsequent nodes for all cell types. Branching children and initial nodes each required additional morphological features to predict diameter, such as path length to soma, total dendritic length, and longest path to terminal end. Model simulations reveal that membrane potential response with predicted diameters is similar to the original response for several tested morphologies. We provide our open source software to extend the utility of available NeuroMorpho.org morphologies, and suggest predictive equations may supplement morphologies that lack dendritic diameter and improve model simulations with realistic dendritic diameter.

Cannabinoid CB1 receptor mediates METH-induced electrophysiological and morphological alterations in cerebellum Purkinje cells.

Our previous studies on cannabinoid type1 receptor (CB1R) activation on Methamphetamine (METH)-induced neurodegeneration and locomotion impairments in male rats suggest an interaction between CB1Rs and METH. However, the role of these receptors in METH-neurotoxicity has not been fully identified. Therefore, the purpose of the present study is to investigate the involvement of CB1Rs in these effects. We conducted an electrophysiological study to evaluate functional interactions between METH and CB1Rs using whole-cell patch current clamp recording. Furthermore, we designed the Nissl staining protocol to assess the effect of METH on the basic cerebellar Purkinje cell structure. Our findings revealed that METH significantly increased the action potential half-width, spontaneous interspike intervals, first spike latency, and decreased the rebound action potential and spontaneous firing frequency. Using CB1R agonist and antagonist, our results showed a significant interaction with some of the electrophysiological alterations induced by METH. Further, Nissl staining revealed that the exposure to the combination of METH and SR141716A resulted in the necrotic cell death. Results of the current study raises the possibility that METH consumption profoundly affect the intrinsic membrane properties of cerebellar Purkinje neurons and cannabinoid system manipulations may counteract some of these effects. In summary, our findings provide further insights into the modulatory role of the endocannabinoid system in METH-induced neurologic changes, which can be used in the development of potential therapeutic interventions for METH dependence.

Multiple signals evoked by unisensory stimulation converge onto cerebellar granule and Purkinje cells in mice

The cerebellum receives signals directly from peripheral sensory systems and indirectly from the neocortex. Even a single tactile stimulus can activate both of these pathways. Here we report how these different types of signals are integrated in the cerebellar cortex. We used in vivo whole-cell recordings from granule cells and unit recordings from Purkinje cells in mice in which primary somatosensory cortex (S1) could be optogenetically inhibited. Tactile stimulation of the upper lip produced two-phase granule cell responses (with latencies of ~8 ms and 29 ms), for which only the late phase was S1 dependent. In Purkinje cells, complex spikes and the late phase of simple spikes were S1 dependent. These results indicate that individual granule cells combine convergent inputs from the periphery and neocortex and send their outputs to Purkinje cells, which then integrate those signals with climbing fiber signals from the neocortex.

BK Channel Gamma3 Subunit (LRRC55) Knock-Out Mice Show Ataxia-Like Phenotype

The large-conductance, calcium- and voltage-activated K+ (BK) channel consists of the pore-forming α subunits (BKα) and the tissue-specific auxiliary subunits. The auxiliary γ3 subunits (LRRC55) modulate the BK channel by shifting the voltage-dependence of channel activation toward the hyperpolarizing direction by approximately 50 mV in the absence of calcium. Previous study showed that LRRC55 is expressed mainly in brains. In this study, we generated LRRC55 knock-out mice and investigated the LRRC55 expression and function in mouse brains. We found that LRRC55 is abundantly expressed in the cerebellum Purkinje cells. LRRC55 knock-out mice performed normally in open-field test, but showed gait abnormality in footprint test. Therefore, deficiency of LRRC55 leads to an ataxia-like phenotype in mice. Our findings suggest an important role of LRRC55 in BK channel regulation in cerebellum.

Quinine associated cerebella ataxia responded with Rehabilitative therapy- A case report from a resource limited country Tanzania

Background Quinine is among the commonly used drugs against malaria and it has been found to have effects against the central nervous system. The most contemporary documented effect is to the cerebellum. Its toxins can affect the layers of the cerebellum molecular (outermost layer), purkinje (middle) and granular layer (innermost). One of the observed effects is ataxia. Despite its effect, it is commonly used in our settings for treatment of complicated malaria. Case presentation A previously well-known 23-years old female, who is a local, second year medical student, presented to the town-based, district-level hospital with a two-day history of headache, joints pain, generalized body malaise, and vomiting. She did not travel to any endemic areas. During her clinical examination, she was noted to be febrile at 38’c. Examination on the central nervous system, cardio respiratory system was unremarkable. Blood slide for malaria parasite revealed 3mps/200wbc. The full blood picture findings were within the normal range except for the granulocytes which were slightly elevated. She was admitted and then injected with 600mg of Quinine (I.V) 8 hourly for 3 days and after she had finished the dose she started swaying with staggering gait. The condition worsened hence leading to a transfer to a resourceful centre for possible Magnetic resonance imaging (MRI). MRI could not depict any lesion in the brain neither the spinal cord. Neuro-rehabilitation was done and eventually after fourteen days the patient recovered. Conclusion Administration of quinine injection may have adverse effects on the postural and balance related structures of the brain. However, the resulting loss of balance and ataxia may resolve with time and with physical therapy. This case report ought to remind medical personnel attending patients especially those suffering from Malaria to be careful and observe the recommended dosage as it may compromise or cause effects to central nervous system. It also reminds urgent referral cases to opt for rehabilitation if undesired effects to the central nervous system occur. Keywords: Quinine, cerebellum, ataxia, rehabilitation.

트레드밀 운동이 노화 흰쥐 소뇌의 성상세포 활성과 퍼킨제 세포 및 운동기능 변화에 미치는 영향

트레드밀 운동이 노화 흰쥐 소뇌의 성상세포 활성과 퍼킨제 세포 및 운동기능 변화에 미치는 영향

Loss of HAX-1 May Contribute to the Neurodegeneration Caused by a Kv3.3 Mutation

Spinocerebellar Ataxia type 13 (SCA13) is caused by mutations in the KCNC3 gene, which encodes the voltage-dependent potassium channel Kv3.3. These channels are expressed at particularly high levels in cerebellum Purkinje cells. SCA13 is an autosomal dominant disease characterized primarily by degeneration of the cerebellum. We have found the Kv3.3 channel differs from other potassium channels in that it binds Hax-1, an anti-apoptotic protein that is absolutely required for the survival of cerebellar neurons. We have also found that depolarization of Kv3.3 channels directly activates Tank Binding Kinase 1 (TBK1), an enzyme that plays a key role in the formation of multivesicular bodies, autophagy and mitophagy. A disease-causing mutation, G592R Kv3.3, produces enhanced TBK1 activation both in cell lines and in the cerebellum of knock-in mice bearing this mutation. By electron microscopy, we find the enhanced activation of TBK1 in G592R Kv3.3 knock-in mice is associated with increased numbers of intracellular multivesicular bodies containing Hax-1, and increased levels of CD63, a molecular marker for these structures. We also found that the expression level of Hax-1 is reduced in G592R Kv3.3 knock-in mice by Western blotting. Mitochondrial function may also be impacted by the mutation because levels of mitofusin-1 and mitofusin-2, proteins that are required for fusion and maintenance of mitochondrial structure, are altered in the mutant mice. Using cell lines expressing the mutant channel, we have shown that the G592R Kv3.3-induced multivesicular bodies contain Hax-1. Inhibition of TBK1 in the cell lines prevents the increase in CD63 levels produced by the Kv3.3 mutation. Our findings suggest that Kv3.3 channels are directly coupled to pathways that regulate the trafficking of proteins into multivesicular bodies and that loss of the Hax-1 may contribute to the neurodegeneration caused by SCA13.

Cостояние митохондрий в клетках Пуркинье мозжечка крысы при холестазе

Cостояние митохондрий в клетках Пуркинье мозжечка крысы при холестазе

Genetic ablation of dynactin p150Glued in postnatal neurons causes preferential degeneration of spinal motor neurons in aged mice

BackgroundDynactin p150Glued, the largest subunit of the dynactin macromolecular complex, binds to both microtubules and tubulin dimers through the N-terminal cytoskeleton-associated protein and glycine-rich (CAP-Gly) and basic domains, and serves as an anti-catastrophe factor in stabilizing microtubules in neurons. P150Glued also initiates dynein-mediated axonal retrograde transport. Multiple missense mutations at the CAP-Gly domain of p150Glued are associated with motor neuron diseases and other neurodegenerative disorders, further supporting the importance of microtubule domains (MTBDs) in p150Glued functions. However, most functional studies were performed in vitro. Whether p150Glued is required for neuronal function and survival in vivo is unknown.MethodsUsing Cre-loxP genetic manipulation, we first generated a line of p150Glued knock-in mice by inserting two LoxP sites flanking the MTBD-coding exons 2 to 4 of p150Glued–encoding Dctn1 gene (Dctn1LoxP/), and then crossbred the resulting Dctn1LoxP/ mice with Thy1-Cre mice to generate the bigenic p150Glued (Dctn1LoxP/LoxP; Thy1-Cre) conditional knockout (cKO) mice for the downstream motor behavioral and neuropathological studies.ResultsP150Glued expression was completely abolished in Cre-expressing postnatal neurons, including corticospinal motor neurons (CSMNs) and spinal motor neurons (SMNs), while the MTBD–truncated forms remained. P150Glued ablation did not affect the formation of dynein/dynactin complex in neurons. The p150Glued cKO mice did not show any obvious developmental phenotypes, but exhibited impairments in motor coordination and rearing after 12 months of age. Around 20% loss of SMNs was found in the lumbar spinal cord of 18-month-old cKO mice, in company with increased gliosis, neuromuscular junction (NMJ) disintegration and muscle atrophy. By contrast, no obvious degeneration of CSMNs, striatal neurons, midbrain dopaminergic neurons, cerebellar granule cells or Purkinje cells was observed. Abnormal accumulation of acetylated α-tubulin, and autophagosome/lysosome proteins was found in the SMNs of aged cKO mice. Additionally, the total and cell surface levels of glutamate receptors were also substantially elevated in the p150Glued-depleted spinal neurons, in correlation with increased vulnerability to excitotoxicity.ConclusionOverall, our findings demonstrate that p150Glued is particularly required to maintain the function and survival of SMNs during aging. P150Glued may exert its protective function through regulating the transportation of autophagosomes, lysosomes, and postsynaptic glutamate receptors in neurons.

Modulation of GABA- and Glycine-Activated Ionic Currents with Semax in Isolated Cerebral Neurons.

The concentration-clamp experiments with neurons isolated from the rat brain showed that nootropic and neuroprotective drug Semax added to perfusion solution at concentration of 1 μM augmented the amplitude of GABA-activated ionic currents in cerebellum Purkinje cells by 147±13%. In addition, Semax in perfusion solution (0.1 and 1 μM) diminished the amplitude of glycine-activated chloride currents in hippocampal pyramidal neurons down to 68 and 43% control level, respectively. Both potentiating and inhibitory effects developed slowly, and they were poorly reversible, which indicated a probable implication of second messengers in the observed phenomena. Semax accelerated the falling edge of glycine-activated current both after a short-term co-application with agonist and after addition of this peptide into perfusion solution.

Key role of external chloride ions in regulation of fast sodium channels in rat cerebellum Purkinje neurons.

A decrease in the external chloride ion concentration to 4 mM caused a decrease in the fast sodium current to 85-100% in rat cerebellum Purkinje neurons in the whole-cell configuration using the patchclamp method. This effect did not depend on the main cation from the internal side of the cell membrane (120 mM of potassium or cesium) and also appeared when Cl- was substituted with sulfate (SO2-4) or phosphate (H2PO4-) anion outside of the cell. The effect was reversible after washing out the low chloride by the standard saline.