Etiological Studies Of Dystonia In Hoxb8 Mutant Rats

Rats have been widely used as one of research animal models in neuroscience and pharmacology,due to advantages such as large size,and physiological and behavioral similarities to human.However,to date,reports using gene-targeting technologies by homologous recombination in rat embryonic stem cells(ES cells)to successfully obtain mutants have been very limited.Here we performed gene targeting in rat ES cells with ACN self-excising cassette,and generated Hoxb8 knockout rats with automatic selection gene removal.The normality of both expression of adjacent genes and skeletal development in Hoxb8 mutants suggested that mutant phenotypes directly resulted from loss-of-fuction of Hoxb8.Through behavioral analyses,we found that Hoxb8-/-rats were not sensitive to nociceptive stimulation,and showed movement disorder with weakness of hindlimb muscle,abnormal posture and movement.In extreme situations,mutant rats exhibited seizure-like events.Nissl staining and immunofluorescence analysis showed that the insensitivity of Hoxb8 mutants to nociceptive stimulation resulted from the distribution and disorganization of sensory neurons in the dorsal spinal cord.Hox genes play leading roles in patterning and development along antero-posterior axis of vertebrate body plans during embryonic development.The most surprising and unexpected phenotype of Hoxb8 mutant mice is that the compulsive grooming behavior of Hoxb8 mutant mice can be rescued by wild-type bone marrow transplantation,establishing a direct link between nervous system and immune system missing in previous studies.However,no reports have shown that obsessive-compulsive disorder(OCD)in human patients is associated with Hoxb8 gene mutation,suggesting that mouse disease models are not able to fully recapitulate human diseases.In addition,dystonia is prevalently present in numerous brain-related autoimmune diseases,such as multiple sclerosis and Sydenham’s chorea.The pathogenesis and pathological characteristics of the neuropsychiatric diseases,stemming from autoimmune activatition of brain antigens are very complicated,and it is difficult to diagnose and study these diseases because of their overlapping symptoms and the separation of blood brain barrier(BBB).Unfortunately,lack of animal models which directly associate neuropsychiatry with autoimmunity,has long been a big obstacle for research in the field.Our establishment of Hoxb8 mutant rat model provided a long sought tool for this sort of research.In this research,we aimed to find possible mechanisms on hindlimb dystonia in Hoxb8-/-rats.We found that the synthesis of catecholamine neurotransmitter dopamine(DA)was disrupted in the central nervous system of Hoxb8-/-rats by governing transcripts of DA synthetase.Unexpected autoantibodies against mouse brain proteins in the sera of Hoxb8-/-rats were detected by Western blot analysis,which indicated the autoimmune system of mutants was activated.Subsequently,dopamine receptors were confirmed as antigens in vitro.These results were also further verified by pharmacological experiments.Dystonia of Hoxb8-/-rats was effectively relieved by intraperitoneal injection of dopamine precursor levodopa(L-DOPA)and dopamine receptor antagonist haloperidol,while the treatment of dopamine receptor agonist apomorphine induced seizures in Hoxb8 mutants.The impairment of dopamine synthesis and the autoantibody activation of dopamine receptors as brain antigen lead to abnormal neurological symptoms in Hoxb8-/-rats,but the mechanismatic relationship between dopamine/dopamine recepors and movement malfunction in Hoxb8 mutants remains unclear.It is necessary to further investigate the mechanism by which how the autoimmune system is activated in Hoxb8 mutant rats.Taken together,Hoxb8 mutant rats were generated in our studies,and exhibited dystonia of hindlimb.Intriguingly,the autoimmune system was activated in mutant rats,manifested by appearance of anti-brain autoantibodies recognizing dopamine receptors as antigens in mutant sera.All of these results suggested that the generation of Hoxb8 mutant rat model perfectly builds the connection between autoimmunity and neuropsychiatry,and provides a new tool to analyse their relationship,explore the pathogenesis and develop new treatment strategies for brain-related autoimmune diseases.