Mutation of POLB Causes Lupus in Mice

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Deletion of RAGE Causes Hyperactivity and Increased Sensitivity to Auditory Stimuli in Mice

The receptor for advanced glycation end-products (RAGE) is a multi-ligand receptor that belongs to the immunoglobulin superfamily of cell surface receptors. In diabetes and Alzheimer''s disease, pathological progression is accelerated by activation of RAGE. However, how RAGE influences gross behavioral activity patterns in basal condition has not been addressed to date. In search for a functional role of RAGE in normal mice, a series of standard behavioral tests were performed on adult RAGE knockout (KO) mice. We observed a solid increase of home cage activity in RAGE KO. In addition, auditory startle response assessment resulted in a higher sensitivity to auditory signal and increased prepulse inhibition in KO mice. There were no significant differences between KO and wild types in behavioral tests for spatial memory and anxiety, as tested by Morris water maze, classical fear conditioning, and elevated plus maze. Our results raise a possibility that systemic therapeutic treatments to occlude RAGE activation may have adverse effects on general activity levels or sensitivity to auditory stimuli.     

A Mild PUM1 Mutation Is Associated with Adult-Onset Ataxia,whereas Haploinsufficiency Causes Developmental Delay and Seizures

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Substance P Causes Seizures in Neurocysticercosis

Neurocysticercosis (NCC), a helminth infection of the brain, is a major cause of seizures. The mediators responsible for seizures in NCC are unknown, and their management remains controversial. Substance P (SP) is a neuropeptide produced by neurons, endothelial cells and immunocytes. The current studies examined the hypothesis that SP mediates seizures in NCC. We demonstrated by immunostaining that 5 of 5 brain biopsies from NCC patients contained substance P (SP)-positive (+) cells adjacent to but not distant from degenerating worms; no SP+ cells were detected in uninfected brains. In a rodent model of NCC, seizures were induced after intrahippocampal injection of SP alone or after injection of extracts of cysticercosis granuloma obtained from infected wild type (WT), but not from infected SP precursor-deficient mice. Seizure activity correlated with SP levels within WT granuloma extracts and was prevented by intrahippocampal pre-injection of SP receptor antagonist. Furthermore, extracts of granulomas from WT mice caused seizures when injected into the hippocampus of WT mice, but not when injected into SP receptor (NK1R) deficient mice. These findings indicate that SP causes seizures in NCC, and, suggests that seizures in NCC in humans may be prevented and/or treated with SP-receptor antagonists.     

A Specific IFIH1 Gain-of-Function Mutation Causes Singleton-Merten Syndrome

Singleton-Merten syndrome (SMS) is an infrequently described autosomal-dominant disorder characterized by early and extreme aortic and valvular calcification, dental anomalies (early-onset periodontitis and root resorption), osteopenia, and acro-osteolysis. To determine the molecular etiology of this disease, we performed whole-exome sequencing and targeted Sanger sequencing. We identified a common missense mutation, c.2465G>A (p.Arg822Gln), in interferon induced with helicase C domain 1 (IFIH1, encoding melanoma differentiation-associated protein 5 [MDA5]) in four SMS subjects from two families and a simplex case. IFIH1 has been linked to a number of autoimmune disorders, including Aicardi-Goutières syndrome. Immunohistochemistry demonstrated the localization of MDA5 in all affected target tissues. In vitro functional analysis revealed that the IFIH1 c.2465G>A mutation enhanced MDA5 function in interferon beta induction. Interferon signature genes were upregulated in SMS individuals’ blood and dental cells. Our data identify a gain-of-function IFIH1 mutation as causing SMS and leading to early arterial calcification and dental inflammation and resorption.     

VAMP1 Mutation Causes Dominant Hereditary Spastic Ataxia in Newfoundland Families

Our group previously described and mapped to chromosomal region 12p13 a form of dominantly inherited hereditary spastic ataxia (HSA) in three large Newfoundland (Canada) families. This report identifies vesicle-associated membrane protein 1 (VAMP1), which encodes a critical protein for synaptic exocytosis, as the responsible gene. In total, 50 affected individuals from these families and three independent probands from Ontario (Canada) share the disease phenotype together with a disruptive VAMP1 mutation that affects a critical donor site for the splicing of VAMP1 isoforms. This mutation leads to the loss of the only VAMP1 isoform (VAMP1A) expressed in the nervous system, thus highlighting an association between the well-studied VAMP1 and a neurological disorder. Given the variable phenotype seen in the affected individuals examined here, we believe that VAMP1 should be tested for mutations in patients with either ataxia or spastic paraplegia.     

The Ataxia (ax J) Mutation Causes Abnormal GABAA Receptor Turnover in Mice

Ataxia represents a pathological coordination failure that often involves functional disturbances in cerebellar circuits. Purkinje cells (PCs) characterize the only output neurons of the cerebellar cortex and critically participate in regulating motor coordination. Although different genetic mutations are known that cause ataxia, little is known about the underlying cellular mechanisms. Here we show that a mutated ax ^J gene locus, encoding the ubiquitin-specific protease 14 (Usp14), negatively influences synaptic receptor turnover. Ax ^J mouse mutants, characterized by cerebellar ataxia, display both increased GABA_A receptor (GABA_AR) levels at PC surface membranes accompanied by enlarged IPSCs. Accordingly, we identify physical interaction of Usp14 and the GABA_AR α1 subunit. Although other currently unknown changes might be involved, our data show that ubiquitin-dependent GABA_AR turnover at cerebellar synapses contributes to ax ^J-mediated behavioural impairment.     

Reduced Life- and Healthspan in Mice Carrying a Mono-Allelic BubR1 MVA Mutation

Mosaic Variegated Aneuploidy (MVA) syndrome is a rare autosomal recessive disorder characterized by inaccurate chromosome segregation and high rates of near-diploid aneuploidy. Children with MVA syndrome die at an early age, are cancer prone, and have progeroid features like facial dysmorphisms, short stature, and cataracts. The majority of MVA cases are linked to mutations in BUBR1, a mitotic checkpoint gene required for proper chromosome segregation. Affected patients either have bi-allelic BUBR1 mutations, with one allele harboring a missense mutation and the other a nonsense mutation, or mono-allelic BUBR1 mutations combined with allelic variants that yield low amounts of wild-type BubR1 protein. Parents of MVA patients that carry single allele mutations have mild mitotic defects, but whether they are at risk for any of the pathologies associated with MVA syndrome is unknown. To address this, we engineered a mouse model for the nonsense mutation 2211insGTTA (referred to as GTTA) found in MVA patients with bi-allelic BUBR1 mutations. Here we report that both the median and maximum lifespans of the resulting BubR1 ^+/GTTA mice are significantly reduced. Furthermore, BubR1 ^+/GTTA mice develop several aging-related phenotypes at an accelerated rate, including cataract formation, lordokyphosis, skeletal muscle wasting, impaired exercise ability, and fat loss. BubR1 ^+/GTTA mice develop mild aneuploidies and show enhanced growth of carcinogen-induced tumors. Collectively, these data demonstrate that the BUBR1 GTTA mutation compromises longevity and healthspan, raising the interesting possibility that mono-allelic changes in BUBR1 might contribute to differences in aging rates in the general population.     

Cardiomyocyte Specific Deletion of Crif1 Causes Mitochondrial Cardiomyopathy in Mice

Mitochondria are key organelles dedicated to energy production. Crif1, which interacts with the large subunit of the mitochondrial ribosome, is indispensable for the mitochondrial translation and membrane insertion of respiratory subunits. To explore the physiological function of Crif1 in the heart, Crif1^f/f mice were crossed with Myh6-cre/Esr1 transgenic mice, which harbor cardiomyocyte-specific Cre activity in a tamoxifen-dependent manner. The tamoxifen injections were given at six weeks postnatal, and the mutant mice survived only five months due to hypertrophic heart failure. In the mutant cardiac muscles, mitochondrial mass dramatically increased, while the inner structure was altered with lack of cristae. Mutant cardiac muscles showed decreased rates of oxygen consumption and ATP production, suggesting that Crif1 plays a critical role in the maintenance of both mitochondrial structure and respiration in cardiac muscles.     

Deletion of PDK1 Causes Cardiac Sodium Current Reduction in Mice

Background

The AGC protein kinase family regulates multiple cellular functions. 3-phosphoinositide-dependent protein kinase-1 (PDK1) is involved in the pathogenesis of arrhythmia, and its downstream factor, Forkhead box O1 (Foxo1), negatively regulates the expression of the cardiac sodium channel, Nav1.5. Mice are known to die suddenly after PDK1 deletion within 11 weeks, but the underlying electrophysiological bases are unclear. Thus, the aim of this study was to investigate the potential mechanisms between PDK1 signaling pathway and cardiac sodium current.

Methods and Results

Using patch clamp and western blotting techniques, we investigated the role of the PDK1-Foxo1 pathway in PDK1 knockout mice and cultured cardiomyocytes. We found that PDK1 knockout mice undergo slower heart rate, prolonged QRS and QTc intervals and abnormal conduction within the first few weeks of birth. Furthermore, the peak sodium current is decreased by 33% in cells lacking PDK1. The phosphorylation of Akt (308T) and Foxo1 (24T) and the expression of Nav1.5 in the myocardium of PDK1-knockout mice are decreased, while the nuclear localization of Foxo1 is increased. The role of the PDK1-Foxo1 pathway in regulating Nav1.5 levels and sodium current density was verified using selective PDK1, Akt and Foxo1 inhibitors and isolated neonatal rat cardiomyocytes.

Conclusion

These results indicate that PDK1 participates in the dysregulation of electrophysiological basis by regulating the PDK1-Foxo1 pathway, which in turn regulates the expression of Nav1.5 and cardiac sodium channel function.     

Hyperphosphorylation and Accumulation of Neurofilament Proteins in Transgenic Mice with Alzheimer Presenilin 1 Mutation

Neurofilaments (NFs) are hyperphosphorylated and accumulate in Alzheimer’s disease (AD) brains. In this study, employing the transgenic mouse model, we explored the effect of presenilin 1 (PS-1) mutation on the phosphorylation and distribution of NFs. Western blot analysis showed that there was a significant increase in the phosphorylation of NF-H and NF-M subunits with a concomitant increase in phosphorylated c-Jun N-terminal protein kinase 1/2 (JNK1/2) mitogen-activated protein kinase (MAPK) in hippocampus of PS-1 transgenic mice compared to that of wild-type littermates. Immunohistochemical analysis revealed that phosphorylated NFs accumulated throughout the hippocampal neurons of the transgenic mice. These findings suggest that PS-1 mutation may induce hyperphosphorylation and accumulation of NFs via a JNK1/2-involved mechanism.     

Loss of Ahi1 Impairs Neurotransmitter Release and Causes Depressive Behaviors in Mice

Major depression is becoming one of the most prevalent forms of psychiatric disorders. However, the mechanisms of major depression are still not well-understood. Most antidepressants are only effective in some patients and produce some serious side effects. Animal models of depression are therefore essential to unravel the mechanisms of depression and to develop novel therapeutic strategies. Our previous studies showed that Abelson helper integration site-1 (Ahi1) deficiency causes depression-like behaviors in mice. In this study, we characterized the biochemical and behavioral changes in Ahi1 knockout (KO) mice. In Ahi1 KO mice, neurotransmitters including serotonin and dopamine were significantly decreased in different brain regions. However, glutamate and GABA levels were not affected by Ahi1 deficiency. The antidepressant imipramine attenuated depressive behaviors and partially restored brain serotonin level in Ahi1 KO mice. Our findings suggest that Ahi1 KO mice can be used for studying the mechanisms of depression and screening therapeutic targets.     

Inhibition of miR-203 Reduces Spontaneous Recurrent Seizures in Mice

Inhibitory synaptic receptors are dysfunctional in epileptic brains, and agents that selectively target these receptors may be effective for the treatment of epilepsy. MicroRNAs interfere with the translation of target genes, including various synaptic proteins. Here, we show that miR-203 regulates glycine receptor-β (Glrb) in epilepsy models. miR-203 is upregulated in the hippocampus of epileptic mice and human epileptic brains and is predicted to target inhibitory synaptic receptors, including Glrb. In vitro transfection, target gene luciferase assays, and analysis of human samples confirmed the direct inhibition of GLRB by miR-203, and AM203, an antagomir targeting miR-203, reversed the effect of miR-203. When intranasal AM203 was administered, AM203 reached the brain and restored hippocampal GLRB levels in epileptic mice. Finally, intranasal AM203 reduced the epileptic seizure frequency of mice. Overall, this study suggests that GLRB expression in the epileptic brain is controlled by miR-203, and intranasal delivery of AM203 showed therapeutic effects in chronic epilepsy mice.     

Inactivation of Uaf1 Causes Defective Homologous Recombination and Early Embryonic Lethality in Mice

The deubiquitinating enzyme heterodimeric complex USP1-UAF1 regulates the Fanconi anemia (FA) DNA repair pathway. Absence of this complex leads to increased cellular levels of ubiquitinated FANCD2 (FANCD2-Ub) and ubiquitinated PCNA (PCNA-Ub). Mice deficient in the catalytic subunit of the complex, USP1, exhibit an FA-like phenotype and have a cellular deficiency in homologous-recombination (HR) repair. Here, we have characterized mice deficient in the UAF1 subunit. Uaf1^+/− mice were small at birth and exhibited reduced fertility, thus resembling Usp1^−/− mice. Unexpectedly, homozygous Uaf1^−/− embryos died at embryonic day 7.5 (E7.5). These mutant embryos were small and developmentally retarded. As expected, Uaf1 deficiency in mice led to increased levels of cellular Fancd2-Ub and Pcna-Ub. Uaf1^+/− murine embryonic fibroblasts (MEFs) exhibited profound chromosome instability, genotoxin hypersensitivity, and a significant defect in homologous-recombination repair. Moreover, Uaf1^−/− mouse embryonic stem cells (mESCs) showed chromosome instability, genotoxin hypersensitivity, and impaired Fancd2 focus assembly. Similar to USP1 knockdown, UAF1 knockdown in tumor cells caused suppression of tumor growth in vivo. Taken together, our data demonstrate the important regulatory role of the USP1-UAF1 complex in HR repair through its regulation of the FANCD2-Ub and PCNA-Ub cellular pools.     

Anticonvulsant Effect of Swertiamarin Against Pilocarpine-Induced Seizures in Adult Male Mice

Epilepsy is one of the common and major neurological disorders, approximately a third of the individuals with epilepsy suffer from seizures and not able to successfully respond to available medications. Current study was designed to investigate whether Swertiamarin (Swe) had anticonvulsant activity in the pilocarpine (PILO)-treated mice. Thirty minutes prior to the PILO (280 mg/kg) injection, the mice were administrated with Swe (50, 150, and 450 mg/kg) and valproate sodium (VPA, 200 mg/kg) once. Seizures and electroencephalography (EEG) were observed, and then the mice were killed for Nissl, Fluoro-jade B (FJB) staining. Astrocytic activation was examined in the hippocampus. Western blot analysis was used to examine the expressions of interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10). The results indicated that pretreatment with Swe (150, 450 mg/kg) and VPA (200 mg/kg) significantly delayed the onset of the first convulsion and reduced the incidence of status epilepticus and mortality. Analysis of EEG recordings demonstrated that Swe (150, 450 mg/kg) and VPA (200 mg/kg) sharply decreased epileptiform discharges. Furthermore, Nissl and FJB staining revealed that Swe (150, 450 mg/kg) and VPA (200 mg/kg) relieved the neuronal damage. Additionally, Swe (450 mg/kg) dramatically inhibited astrocytic activation. Western blot analysis showed that Swe (450 mg/kg) significantly decreased the expressions of IL-1β, IL-6, TNF-α and elevated the expression of IL-10. Taken together, these findings revealed that Swe exerted anticonvulsant effects on PILO-treated mice. Further studies are encouraged to investigate these beneficial effects of Swe as an adjuvant in epilepsy.     

POC1A Truncation Mutation Causes a Ciliopathy in Humans Characterized by Primordial Dwarfism

Primordial dwarfism (PD) is a phenotype characterized by profound growth retardation that is prenatal in onset. Significant strides have been made in the last few years toward improved understanding of the molecular underpinning of the limited growth that characterizes the embryonic and postnatal development of PD individuals. These include impaired mitotic mechanics, abnormal IGF2 expression, perturbed DNA-damage response, defective spliceosomal machinery, and abnormal replication licensing. In three families affected by a distinct form of PD, we identified a founder truncating mutation in POC1A. This gene is one of two vertebrate paralogs of POC1, which encodes one of the most abundant proteins in the Chlamydomonas centriole proteome. Cells derived from the index individual have abnormal mitotic mechanics with multipolar spindles, in addition to clearly impaired ciliogenesis. siRNA knockdown of POC1A in fibroblast cells recapitulates this ciliogenesis defect. Our findings highlight a human ciliopathy syndrome caused by deficiency of a major centriolar protein.     

TNNI2突变转基因小鼠模型的建立

目的建立TNNI2突变转基因小鼠模型。方法构建pEGFP-tnni2转基因构件,TNNI2基因的第175个氨基酸缺失,通过原核显微注射法。把线性化、纯化后的外源基因pEGFP-tnni2注射入BDF1小鼠受精卵中。胚胎移植给同期发情的假孕受体母鼠,获得子代小鼠。用PCR和Southern方法检测子代鼠尾DNA鉴定基因型。通过RT-PCR方法检测tnni2基因表达。结果移植注射胚胎115枚给4只假孕小鼠共出生了23只后代鼠。经PCR和Southern方法检测得到4只阳性小鼠。对其子代进行RT-PCR检测,tnni2基因在肌肉、心脏内表达。结论通过显微注射法使外源基因pEGFP-tnni2（TNNI2基因的第175个氨基酸缺失）在小鼠基因组中得到整合,建立了转pEGFP-tnni2的转基因小鼠模型。