Nipbl Maintains Postnatal CA1 Hippocampus Integrity

Background and aims:Cornelia de Lange syndrome(Cd LS)(Online Mendelian Inheritance in Man(OMIM)entries 122470,300590,300882,610759 and 614701)is a MCA/MR(multiple congenital anomalies/mental retardation)condition.Its clinical presentation is characterized by facial dysmorphism,prenatal and postnatal growth retardation,cognitive impairment ranging from mild to severe,gastrointestinal malformations,congenital heart abnormalities and limb defects.Pathogenic genetic variants encoding cohesion complex subunits and interacting proteins(e.g.,NIPBL,SMC1 A,SMC3,HDAC8,and RAD21)are the major causes of Cd LS.Cohesin is an essential regulator of most aspects of chromosome biology,including chromosome segregation,maintenance of genome stability,regulation of gene expression,chromatin structure and genome organization.Although the exact pathomechanisms in Cd LS are currently not fully understood,the direct role of cohesin as a regulator of gene expression is estimated to be of crucial importance for cohesin function.Heterozygous mutations in the cohesin loading factor Nipped-B-like(NIPBL)have been identified in 50%?60% of cases and NIPBL expression levels in Cd LS probands predict phenotypic severity.Although the genetic basis of Cd LS is now uncovered,how behavioral manifestations are associated with genetic and brain differences is still yet to be elucidated.Recent work has uncovered that depletion of Nipbl from cortical neural progenitors in vivo is detrimental to neuronal migration,but the brain-region-specific contributions of Nipbl to intellectual disability and autism phenotypes have yet to be determined fully.Here,we describe NIPBL conditional knock-out(NIPBL c KO)male and female mice with loss of Nipbl in the pyramidal neurons of layer V cerebral cortex and the CA1 subfield of the hippocampus.Methods:Layer V cerebral cortex and CA1 hippocampus excitatory glutamatergic neuron-specific KO mice for Nipbl were created by breeding Ca MKII?-Cre transgenic mice line with Nipbl-Lox P mice generated previously.These animals have one or two Ca MKII?-Cre alleles and are designated NIPBL c KO(-/-)at ?p7.They consistently show the same phenotypes with high penetrance.Only c KO animals were used for this study.All mice were genotyped by PCR before experimentation.Genotypes,as well as knock-out of NIPBL,were confirmed by PCR.For all experiments,WT(+/+)were used as controls.We characterize mice NIPBL c KO mice in a number of behavioral paradigms,including the open field test,the Morris water maze(MWM)and the nest building test.After behavioral testing was completed,we compared brain area between littermate homozygous mutant and WT animals to assess neuroanatomical alterations due to forebrain Nipbl loss that could explain the observed phenotypes.Immunofluorescence of the dendritic protein MAP2 were performed in the cortex and hippocampus of 6-month-old mice to determine if there are any fine morphological changes in the dendritic or synaptic morphology at this time point.Terminal deoxynucleotidyl transferase d UTP nick end labeling(TUNEL)was used to assess apoptosis in the cortex and hippocampus.To determine the processes governing the behavioral phenotypes in NIPBL c KO mice,we ascertained transcriptional changes due to Nipbl loss by carrying out RNA-seq on tissue samples from the hippocampus of both NIPBL c KO mice and littermate controls.Results:1.NIPBL m RNA was reduced in NIPBL c KO mouse hippocampus and cerebral cortex,both brain regions with high Camk2a-Cre activity.2.Overall,NIPBL c KO mice showed postnatal growth retardation and early lethality.3.Comparing to WT mice,NIPBL c KO mice exhibited significantly increased exploratory behaviors in the open-field test,as demonstrated by the increased time spent in the center compartment [WT: 86.95±11.56 sec;NIPBL c KO: 151.6±24.61 sec,n=8–11 per group,t(17)=2.603,p=0.0186,t test].The increased exploratory behaviors in NIPBL c KO mice were not due to hyperactivity,as total distance covered was not significantly different between genotypes [WT: 4437±181;NIPBL c KO: 5668±724.6,n=8–11 per group,t(17)=1.901,p=0.0744,t test].4.Analysis of the data from the place trials showed robust acquisition deficits in the NIPBL c KO mice compared with the control group in terms of path length,(genotype effect: **** p<0.0001).Importantly,the control group showed a significant decrease in path length across blocks of trials(block 1 vs block 5: ****p<0.0001),whereas the NIPBL c KO mice did not,but rather exhibited similar path lengths throughout acquisition training,suggesting that the control mice learned the location of X the hidden platform whereas the NIPBL c KO mice failed to do so.Similar findings were observed with regard to escape latency.Consistent with the place learning results,the retention performance of the NIPBL c KO mice was impaired profoundly across two dependent variables analyzed from the probe trial.Specifically,the NIPBL c KO group made significantly fewer platform crossings [t(16)=6.205: p<0.0001] and spent significantly less time in the target quadrant [t(16)=3.063: p=0.0074] compared with controls.In summary,the 6-to 8-week-old NIPBL c KO mice exhibited severe spatial learning and memory impairments to the degree that there was no evidence that any learning had occurred in these mutant mice.5.NIPBL c KO animals consistently produced low-quality nests and,in most cases,never interacted with the provided nesting material [t(14)=39,**** p<0.0001].6.NIPBL mutant mice show neocortical lamination that is largely indistinguishable from wild type and show normal patterns of neocortical neurogenesis and neuronal migration.In contrast,the hippocampus of NIPBL c KO mice shows disrupted lamination that is most severe in the CA1 region.7.Microtubule-associated protein 2(MAP-2)staining revealed that the dendritic arbors in the hippocampus CA1 region of NIPBL c KO were thin,tortuous,and fragmented,consistent with our observations of dendritic arbors in Cd LS brain.However,the structure of the dendrites of cortical neurons was relatively preserved.8.TUNEL analysis revealed no increased apoptosis in NIPBL c KO mice.9.In terms of gene expression,175 transcripts were expressed at significantly different levels between Nipbl-deficient and control hippocampus.Gene Ontology analysis for biological process revealed that dysregulated genes were enriched in categories including axon part,axon terminus,neuron projection terminus and vesicle.Conclusions:1.We show that loss of Nipbl in excitatory glutamatergic neurons of layer V cerebral cortex and CA1 hippocampus is sufficient to produce ASD-and ID-relevant behaviors such as decreased sociability,and impaired spatial learning and memory.2.In addition,we show that the ID-like learning and memory deficits observed in NIPBL c KO mice are likely due to alterations in hippocampal function.CA1 pyramidal neurons from mice postnatally lacking Nipbl exhibit dendritic/synaptic pathologies,and undergo dispersion independently of neuronal migration defect.3.Thus,Nipbl contribute to maintain postnatal CA1 integrity.4.Using RNA-sequencing(RNA-seq),we correlate these behavioral phenotypes to specific changes in the transcriptome of the NIPBL c KO hippocampus.5.As a whole,our data suggest that certain behavioral consequences of Nipbl loss can be attributed to disrupted gene networks within distinct regions of the brain.Therefore,these data could lead to improved treatments for specific ASD and ID symptoms.Our data provide a rationale for the neurological defects associated with deleterious variants in NIPBL and may also serve as a basis for understanding the mechanisms underlying postnatal hippocampal maintenance and function.