Objective: To investigate the effects of RNA m6A demethylase ALKBH5 gene deficiency on cerebellar morphology and function in the aged mice, and to explore the role of ALKBH5 in cerebellar degeneration. Methods: Western blot was performed to detect the protein level of ALKBH5 in the cerebellum of wild-type mice of various ages. The expression of NeuN, Calbindin-D28K, MAP2, GFAP and other proteins in the cerebella of middle-aged (12-month-old) and aged (18-month-old) wild-type mice and ALKBH5^-/- mice was examined using immunohistochemistry. The balance beam test and gait analysis were performed to test the balance ability and motor coordination of the mice. Results: With aging of the mice, the expression of ALKBH5 in the cerebellum increased gradually in an age-dependent manner. In the aged mice, but not middle-aged mice, the body weight, whole brain weight and cerebellum weight of ALKBH5^-/- mice decreased by 15%, 10% and 21%, respectively (P<0.05). The expression of ALKBH5 in the Purkinje cells was much higher than that in other types of neural cells. Correspondingly, ALKBH5-deficiency caused 40% reduction in the number of Purkinje cells, as well as the length and density of neuronal dendrites in the aged mice (P<0.01). In addition, the time for the aged ALKBH5^-/- mice to pass the balance beam was 70% longer than that of the wild type mice of the same age, with unstable gaits (P<0.01). Conclusions: Gene deficiency of RNA m6A demethylase ALKBH5 causes cerebellar atrophy, Purkinje neuron loss and damage in the aged mice. These changes eventually affect mice's motor coordination and balance ability. These results suggest that imbalanced RNA m6A methylation may lead to neurodegenerative lesions in the cerebellum of mice.
Animals ; Mice ; AlkB Homolog 5, RNA Demethylase/metabolism* ; Cerebellum/metabolism* ; Methylation ; RNA/metabolism*

OBJECTIVETo investigate the growth and development of nerve growth factor (NGF)-positive neurons in the cerebellum of midanaphase human fetus.

METHODSThe expression of the NGF-positive neurons in the cerebrum of human fetus was observed by immunohistochemical methods, and the integral absorbance (IA) was detected.

RESULTSBy the 3rd to 4th month of gestation, neurons was seen in the ependymal, central, and marginal plate of cerebellum; the nucleus was oval and the neurons had short and small processes. By the 5th to 7th month of gestation, the number of NGF-positive neurons increased, the expressions enhanced, the nucleus was round-, oval-, or fusiform-shaped, the neurons grew larger in size, and the Purkinje cells showed NGF-positive expression. By the 8th to 10th month of gestation, the NGF-positive expression was enhanced with deeper dying, the body of Purkinje cells grew larger gradually, and the number of NGF-positive neurons in the granular cell layer and molecular layer increased. IA of the cerebellar cortical neurons of the 3rd, 4th, 5th, 6th, 7th, and 8th month of gestation showed an increasing trend, and significant difference was observed (P < 0. 05).

CONCLUSIONNGF-positive neurons in the cerebellum play an important role for differentiation, proliferation, migration, and growth of neurons in the cerebellum.

Cerebellum ; cytology ; metabolism ; Fetus ; cytology ; metabolism ; Humans ; Nerve Growth Factor ; metabolism ; Neurons ; cytology ; metabolism ; Purkinje Cells ; metabolism

Secretion of neurotransmitters is initiated by voltagegated calcium influx through presynaptic, voltage- gated N-type calcium channels. However, little is known about their cellular distribution in the mouse cerebellum. In the cerebellum, alpha1B immunoreactivity is found mainly on the cell bodies of all Purkinje cells. In addition, the immunoreactivity was detected on a subset of Purkinje cell dendrites, clustered to form a parasagittal array of bands. In the anterior lobe vermis, immunoreactive Purkinje cell dendrites form narrow stripes separated by broad bands of unstained dendrites. Moving caudally through the vermis, these stripes become thicker as a larger fraction of the Purkinje cell dendrites become immunoreactive. This localization study of the alpha1B pore-forming subunits in mouse cerebellum may guide future investigations of the role of calcium channels in neurological pathways.
Animals ; Calcium Channels, N-Type/*metabolism ; Cerebellum/cytology/*metabolism ; Dendrites/metabolism ; Immunohistochemistry ; Mice ; Mice, Inbred BALB C ; Purkinje Cells/metabolism

The Pogo mouse is an autosomal recessive ataxic mutant that arose spontaneously in the inbred KJR/MsKist strain derived originally from Korean wild mice. The ataxic phenotype is characterized by difficulty in maintaining posture and side to side stability, faulty coordination between limbs and trunk, and the consequent inability to walk straight. In the present study, the cerebellar concentrations of glutamate and GABA were analyzed, since glutamate is a most prevalent excitatory neurotransmitter whereas gammar-aminobutyric acid (GABA) is one of the most abundant inhibitory neurotransmitters, which may be the main neurotransmitters related with the ataxia and epilepsy. The concentration of glutamate of cerebellum decreased significantly in ataxic mutant Pogo mouse compared to those of control mouse. However, GABA concentration was not decrease. These results suggested that the decrease in glutamate concentration may contribute to ataxia in mutant Pogo mouse.
Animals ; Calcium-Binding Protein, Vitamin D-Dependent/metabolism ; Cerebellum/*metabolism/pathology ; Gait Ataxia/*metabolism/pathology ; Glutamic Acid/*metabolism ; Immunohistochemistry ; Mice ; Mice, Mutant Strains ; gamma-Aminobutyric Acid/*metabolism

Cerebellar Purkinje cells (PCs) exhibit two types of discharge activities: simple spike (SS) and complex spike (CS). Previous studies found that noradrenaline (NA) can inhibit CS and bidirectionally regulate SS, but the enhancement of NA on SS is overwhelmed by the strong inhibition of excitatory molecular layer interneurons. However, the mechanism underlying the effect of NA on SS discharge frequency is not clear. Therefore, in the present study, we examined the mechanism underlying the increasing effect of NA on SS firing of PC in mouse cerebellar cortex in vivo and in cerebellar slice by cell-attached and whole-cell recording technique and pharmacological methods. GABA_A receptor was blocked by 100 µmol/L picrotoxin in the whole process. In vivo results showed that NA significantly reduced the number of spikelets of spontaneous CS and enhanced the discharge frequency of SS, but did not affect the discharge frequency of CS. In vitro experiments showed that NA reduced the number of CS spikelets and after hyperpolarization potential (AHP) induced by electrical stimulation, and increased the discharge frequency of SS. NA also reduced the amplitude of excitatory postsynaptic current (EPSC) of parallel fiber (PF)-PC and significantly increased the paired-pulse ratio (PPR). Application of yohimbine, an antagonist of α2-adrenergic receptor (AR), completely eliminated the enhancing effect of NA on SS. The α2-AR agonist, UK14304, also increased the frequency of SS. The β-AR blocker, propranolol, did not affect the effects of NA on PC. These results suggest that in the absence of GABA_A receptors, NA could attenuate the synaptic transmission of climbing fiber (CF)-PC via activating α2-AR, inhibit CS activity and reduce AHP, thus enhancing the SS discharge frequency of PC. This result suggests that NA neurons of locus coeruleus can finely regulate PC signal output by regulating CF-PC synaptic transmission.
Action Potentials/physiology* ; Animals ; Cerebellar Cortex/metabolism* ; Cerebellum/metabolism* ; Mice ; Norepinephrine/pharmacology* ; Purkinje Cells/metabolism* ; Receptors, Adrenergic, alpha-2/metabolism* ; Receptors, GABA-A/metabolism*

OBJECTIVETo investigate the presence of β-amyloid peptide (Aβ) deposition in the cerebellum and the expression of related miRNAs in the cerebellum of a mouse model of Alzheimer disease.

METHODSTwelve 12-month-old APPswe/PSδE9 double transgenic mice and 12 wild-type C57 mice were sacrificed and the brain tissues were taken for examination. The right hemisphere was stained with Congo red to observe the deposition of amyloid substances, and from the left hemisphere, the hippocampus and the cerebellum were dissected for detecting the expression of miRNA-135a-5p, miRNA-298-5p, miRNA-466b-3p and miR-669f-3p using real-time PCR.

RESULTSCongo red staining revealed the presence of Aβ deposition in both the hippocampus and the cerebellum of the transgenic mice but not in the control mice. Real-time PCR showed a significantly lower expression of the 4 miRNAs in the hippocampus in the transgenic mice than in the control mice (P<0.05). The expression of miRNA-135a-5p, miRNA-298-5p, and miR-669f-3p in the cerebellum was significantly lower in the transgenic mice than in the control mice (P<0.05). The expression of miRNA-298-5p and miR-669f-3p in the hippocampus was significantly lower than that in the cerebellum of the transgenic mice (P<0.05).

CONCLUSIONβ deposition also occurs in the cerebellum of APPswe/PSδE9 double transgenic mice, and its formation might be related to the down-regulation of miRNA-135a-5p, miRNA-298-5p, and miR-669f-3p.

Alzheimer Disease ; metabolism ; Amyloid beta-Peptides ; metabolism ; Animals ; Cerebellum ; metabolism ; Disease Models, Animal ; Hippocampus ; metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; MicroRNAs ; metabolism

OBJECTIVETo detect the expression of signal transducer and activator of transcription 3 (STAT3) and P-STAT3 in the brain of the APPswe/PS δE9 double transgenic mouse model of Alzhaimer's disease (AD) and investigate their possible role in AD.

METHODSAPPswe/PS δE9 double transgenic mice and control mice were examined for cerebral STAT3 and P-STAT3 expressions using immunothistochemistry.

RESULTSSTAT3 and P-STAT3 were expressed in the different regions of mouse brain. In the transgenic mice and the control mice, the positivity rates of STAT3 were 93.75% and 87.50% in the cerebral cortex, 87.50% and 43.75% in the basal forebrain, 81.25% and 37.50% in the hippocampus, and 62.50% and 0.00% in the cerebellum, respectively, showing significant differences between the mice in the STAT3 expressions in the basal forebrain, hippocampus and cerebellum (P<0.05). The positivity rates of P-STAT3 in the two groups were 0.00% and 0.00% in the cerebral cortex, 68.75% and 0.00% in the basal forebrain, 62.50% and 12.50% in the hippocampus, and 43.75% and 0.00% in the cerebellum, respectively, showing also significant differences in the basal forebrain, hippocampus and cerebellum (P<0.05). The expression of STAT3 was positively correlated with that of P-STAT3 in transgenic AD mice (P<0.05).

CONCLUSIONSTAT3 and P-STAT3 are highly expressed in the basal forebrain, hippocampus and cerebellum in transgenic AD mice and may participate in the pathological process of AD.

Alzheimer Disease ; metabolism ; Animals ; Cerebellum ; metabolism ; Cerebral Cortex ; metabolism ; Disease Models, Animal ; Hippocampus ; metabolism ; Mice ; Mice, Transgenic ; STAT3 Transcription Factor ; metabolism

OBJECTIVE: To observe the expression of Muscarinic receptor M1, M3, M5 subunits in rat flocculus following left unilateral labyrinthectomy (UL). METHOD: The RT-PCR was used to observe the expression of Muscarinic receptor M1, M3, M5 subunits post-unilateral labyrinthectomy and investigate its effect on vestibular compensation. RESULT: Muscarinic receptor M1, M3, M5 subunits were induced decrease in both side flocculus after unilateral labyrinthectomy. The expression was the least in the 1 d flocculus of following UL. The expression is rising from the 3-7 d flocculus of following UL. No difference was observed in the 7 d and sham operation flocculus following UL. No difference was observed in the ipsilateral and contralateral flocculus at any group. CONCLUSION Muscarinic receptor M1, M3, M5 subunits were induced decrease in the flocculus after unilateral labyrinthectomy. But the significance of the change of Muscarinic receptor M1, M3, M5 subunits in the vestibular compensation is still unknown.
Animals ; Cerebellum ; metabolism ; Functional Laterality ; Gene Expression ; Male ; Postoperative Period ; Rats ; Receptor, Muscarinic M1 ; metabolism ; Receptor, Muscarinic M3 ; metabolism ; Receptor, Muscarinic M5 ; metabolism ; Vestibule, Labyrinth ; metabolism ; surgery

Mitochondrial calcium uniporter (MCU) is a conserved Ca(2+) transporter at mitochondrial in eukaryotic cells. However, the role of MCU protein in oxidative stress-induced cell death remains unclear. Here, we showed that ectopically expressed MCU is mitochondrial localized in both HeLa and primary cerebellar granule neurons (CGNs). Knockdown of endogenous MCU decreases mitochondrial Ca(2+) uptake following histamine stimulation and attenuates cell death induced by oxidative stress in both HeLa cells and CGNs. We also found MCU interacts with VDAC1 and mediates VDAC1 overexpression-induced cell death in CGNs. This finding demonstrates that MCU-VDAC1 complex regulates mitochondrial Ca(2+) uptake and oxidative stress-induced apoptosis, which might represent therapeutic targets for oxidative stress related diseases.
Animals ; Apoptosis ; Biological Transport ; Calcium ; metabolism ; Calcium Channels ; metabolism ; Cerebellum ; cytology ; HeLa Cells ; Humans ; Mice ; Mitochondria ; metabolism ; Neurons ; cytology ; metabolism ; Oxidative Stress ; Voltage-Dependent Anion Channels ; metabolism

This study was designed to elucidate the existence of PSD95 in the rat sciatic nerve. Immunohistochemical stains of cryosection and teased fiber of sciatic nerves were performed with goat polyclonal antibody against PSD95. Western blot analysis was also accomplished with the same antibody. We got an interesting result that the rat sciatic nerve obviously showed PSD95 immunoreactivity especially in the nodal and paranodal regions, and we also identified a distinct band of PSD95 by western blot. These results suggest PSD95 exists in the sciatic nerve as well as it does in the central nervous system. We suppose PSD95 may have some important roles in ion channel clustering, junctional plasticity and signal transduction in the peripheral nerves as well.
Animals ; Blotting, Western ; Cerebellum/cytology/metabolism ; Immunohistochemistry ; Nerve Fibers/metabolism/ultrastructure ; Nerve Tissue Proteins/*metabolism ; Rats ; Rats, Sprague-Dawley ; Sciatic Nerve/*metabolism