Generation And Characterization Of A Mouse Model For Idiopathic Basal Ganglia Calcification

Idiopathic basal ganglia calcification（IBGC）is an inherited neurodegenerative disorder characterized by progressive calcification deposits in the bilateral brain,which always accompany with dystonia,ataxia,Parkinson-like symptoms,dementia,depression,headaches,epilepsy,and other symptoms.SLC20A2 as the first identified pathogenic gene of IBGC is reported by our research group in 2012.SLC20A2 encodes the type III sodium-dependent phosphate transporter 2（PiT2）,which mainly transports the extracellular Pi into the intracellular for maintaining phosphate homeostasis.IBGC probably resulted from extracellular Pi accumulation by loss of Pi transport function in SLC20A2 mutations.Studies have shown that SLC20A2 is the major pathogenic gene of IBGC with a mutation frequency of 40-50%.However,the mechanism of IBGC resulted from SLC20A2 is largely unknown,and no effective treatment program for IBGC has been reported.In order to explore the mechanism and potentially preventive and therapeutic strategy for IBGC,an appropriate animal model of IBGC is necessary.Currently,mouse has been a perferred mammal model for exploring the function of pathogenic genes of human diseases,due to that the tissue,organ and structure of mouse are similar to that of human and the gene-editing technologies of mouse have been mature enough.In addition,the PiT2 of mouse and human present an identity of 91%and a similar expression in nervous system.Therefore,we generated the Slc20a2 knock-in mouse carrying the mutant site of Slc20a2 p.S602W,which was the allelic ortholog site of human SLC20A2 p.S601W.Detection of 10-month-old wild-type（WT）,heterozygous Slc20a2 knock-in(Slc20a2^+/S602W)and homozygous Slc20a2 knock-in(Slc20a2^S602W/S602W)mice,only Slc20a2^S602W/S602W mice showed severe brain calcification.Further detection of Slc20a2^S602W/S602W mice at 2,2.5,3,4,6 and 8 months old,the calcified nodules with a small size were first observed in thalamus at 3 months old;at the age of 4 months old,brain calcification became obvious,which mainly distributed in thalamus;after 6 months old,brain calcification could be clearly observed in thalamus,striatum,hypothalamus,midbrain,cortex and hippocampus,and calcification in thalamus was most severe.The above results demonstrated that the occurrence and development of brian calcificication of IBGC was a progressive process with age.Submicroscopic structure of brain calcification showed that calcified nodules were varied in size and shape and had a concentric-layer-like structure with alternating high-and medium-density loops.IBGC patients always accompany with extrapyramidal symptoms due to the progressive brain calcification with age,whether Slc20a2^S602W/S602W mice present similar behaviors?Behavior tests showed that Slc20a2^S602W/S602W mice had worse motor balance and coordination,reduced anxiety-like responses and poorer memory performance than WT mice at 14 months old.These studies indicatied that Slc20a2^S602W/S602W mouse was a good IBGC model,which had successfully simulated clinical symptoms of IBGC patients.In addition,similar to IBGC patients,Slc20a2^S602W/S602W mice showed normal growth and development,which would contribute to the rapid propagation of mice to speed up the progress of the related research and be clear to observe side effects of mice in drug research.In previous studies in vitrio from our research group,SLC20A2 mutations were identified destroying Pi transport activity.Hence,we speculated that SLC20A2 mutations would lead to extracellular Pi accumulation and cause a partial increase of Pi concentration,which further resulted in brain calcification.To verify the above speculation in vivo,detection of Pi level in cerebrospinal fluid（CSF）and serum was performed in WT,Slc20a2^+/S602W and Slc20a2^S602W/S602W mice at 2 months old by molybdate-malachite green spectrophotometry.Slc20a2^+/S602W mice showed a remarkable increased CSF Pi level（1.858 mM）but Slc20a2^+/S602W mice showed a normal CSF Pi level（1.163 mM）compared with that of WT mice（1.149 mM）.In addition,serum Pi level showed no change among WT（3.961 mM）,Slc20a2^+/S602W（3.996 mM）and Slc20a2^S602W/S602W（3.857 mM）mice.These results indicated that PiT2 palyed an important role in maintaining CSF Pi homeostasis and the increased CSF Pi level might be the pathophysiological basis of brain calcification.To further reveal the mechanism of IBGC resulting from SLC20A2 mutations,isobaric tags for relative and absolute quantitation（iTRAQ）analysis of brain identified that osteopontin（encoded by Spp1）,osteocalcin（encoded by Mgp）and alpha-2 chain of type IV collagen（encoded by Col4a2）were raised both in Slc20a2^S602W/S602W and homozygous Slc20a2 knockout(Slc20a2^-/-)mice compared with WT mice.By real-time PCR assessment,the mRNA levels of Spp1,Mgp and Col4a2 were also upregulated in thalamus of Slc20a2^S602W/S602W mice compared with WT mice.The serial sections that one for HE staining for calcification and another adjacent one for immunofluorescence staining showed that both osteopontin and type IV collagen were colocalized with brain calcification in Slc20a2^S602W/S602W mice,demonstrating that these proteins were parts of organic components of brain calcification.Meanwhile,the osteopontin level of CSF was quantified by an Elisa kit and was significantly increased in Slc20a2^S602W/S602W mice compared with WT mice at 3 months old,indicating that osteopontin might be a protein marker of brain calcification.Further studies with histology showed that amounts of calcified nodules with a small size were closely connected to blood vessels and amounts of extracellular vesicles were existed around calcified nodules.Detection of the proteins associated with vascular calcification were performed,we found that both alkaline phosphatase and osteoprotegerin were colocalized with brain calcification.Previous studies have shown that the physiological Pi/pyrophosphoric acid（PPi）ratio is a critical indicator for preventing ectopic calcification.Pi is a pro-calcification factor but pyrophosphoric acid（PPi）acts as an anti-calcification role.For exploring potentially preventive and therapeutic strategy for IBGC,we try to alter the PPi or Pi level in the brain of IBGC mouse model.As we know,bisphosphonate drug,having an analogue structure of PPi,is widely used in the studies of treatment exploration of ectopic calcification.Here,etidronate disodium,a kind of bisphosphonate drug,was provided by oral administration for Slc20a2^S602W/S602W mice after weaning.Compared with normal drinking water group of Slc20a2^S602W/S602W mice,etidronate disodium group of Slc20a2^S602W/S602W mice showed no difference on the grading of brain calcification at 4,5,6 and 7 months old,indicating that etidronate disodium could not inhibit brain calcification in Slc20a2^S602W/S602W mice.Meanwhile,a low-phosphate diet was fed to Slc20a2^-/-mice for 1 month.In non-calcified Slc20a2^-/-mice at 2 months old,a low-phosphate diet could decrease the elevated CSF Pi level to normal and effectively inhibit the formation of brain calcification.In calcified Slc20a2^-/-mice at 3 months old,a low-phosphate diet could partially decrease the elevated CSF Pi level and delay the speed of calcification accumulation.However,a long-term low-phosphate diet would decrease serum Pi level and finaly result in hypophosphatemia.For all this,the study of a low-phosphate diet inhibiting brain calcification have provided great contribution to the understanding of IBGC pathogenesis and the prevention and treatment of IBGC.In this thesis,we successfully generated an IBGC model of mouse which closely simulated the pathologic and clinical features of IBGC.To study the mechanism of brain calcification in Slc20a2^S602W/S602W mice,the formation of brain calcification was associatied with cerebral Pi dyshomeostasis.Meanwhile,osteopontin,type IV collagen,alkaline phosphatase and osteoprotegerin were first demonstrated to be associated with brain calcification and be parts of organic components of brain calcification.In addition,the increased osteopontin level in CSF might also be an indicator of brain calcification.To study the preventive and therapeutic strategy of brain calcification in IBGC mouse model,etidronate disodium was verified no efficacy for inhibiting brain calcification.However,a low-phosphate diet could effectively inhibit the formation of brain calcification and delay the speed of calcification accumulation in Slc20a2^-/-mice.The results of these studies have enriched the IBGC models of animal,promoted better understanding for the mechanism of IBGC and contributed to the study of preventive and therapeutic strategy for IBGC.