In early stages of Alzheimer’s disease (AD), neurofibrillary tangles (NFT) are largely restricted to the entorhinal cortex and medial temporal lobe. At later stages, when clinical symptoms generally occur, NFT involve widespread limbic and association cortices. At this point in the disease, amyloid plaques are also abundantly distributed in the cortex. This observation from human neuropathological studies led us to pose two alternative hypotheses: that amyloid in the cortex is permissive for the spread of tangles from the medial temporal lobe, or that these are co-occurring but not causally related events simply reflecting progression of AD pathology. We now directly test the hypothesis that cortical amyloid acts as an accelerant for spreading of tangles beyond the medial temporal lobe. We crossed rTgTauEC transgenic mice that demonstrate spread of tau from entorhinal cortex to other brain structures at advanced age with APP/PS1 mice, and examined mice with either NFTs, amyloid pathology, or both. We show that concurrent amyloid deposition in the cortex 1) leads to a dramatic increase in the speed of tau propagation and an extraordinary increase in the spread of tau to distal brain regions, and 2) significantly increases tau-induced neuronal loss. These data strongly support the hypothesis that cortical amyloid accelerates the spread of tangles throughout the cortex and amplifies tangle-associated neural system failure in AD.

Shamil Sunyaev, David Beier and colleagues report an analysis of the fitness effects of heterozygous protein-truncating variants from the Exome Aggregation Consortium. They find that high heterozygous selection coefficients are enriched in Mendelian disease-associated genes and essential mouse genes, suggesting that this coefficient can be used to prioritize candidate disease-associated genes from clinical exome-sequencing data. The evolutionary cost of gene loss is a central question in genetics and has been investigated in model organisms and human cell lines1,2,3. In humans, tolerance of the loss of one or both functional copies of a gene is related to the gene's causal role in disease. However, estimates of the selection and dominance coefficients in humans have been elusive. Here we analyze exome sequence data from 60,706 individuals4 to make genome-wide estimates of selection against heterozygous loss of gene function. Using this distribution of selection coefficients for heterozygous protein-truncating variants (PTVs), we provide corresponding Bayesian estimates for individual genes. We find that genes under the strongest selection are enriched in embryonic lethal mouse knockouts, Mendelian disease-associated genes, and regulators of transcription. Screening by essentiality, we find a large set of genes under strong selection that are likely to have crucial functions but have not yet been thoroughly characterized.

The ciliary kinase NEK8 plays a critical role in situs determination and cystic kidney disease, yet its exact function remains unknown. In this study, we identify ANKS6 as a target and activator of NEK8. ANKS6 requires NEK8 for localizing to the ciliary inversin compartment (IC) and activates NEK8 by binding to its kinase domain. Here we demonstrate the functional importance of this interaction through the analysis of two novel mouse mutations, Anks6^Streaker and Nek8^Roc. Both display heterotaxy, cardiopulmonary malformations and cystic kidneys, a syndrome also characteristic of mutations in Invs and Nphp3, the other known components of the IC. The Anks6^Strkr mutation decreases ANKS6 interaction with NEK8, precluding NEK8 activation. The Nek8^Roc mutation inactivates NEK8 kinase function while preserving ANKS6 localization to the IC. Together, these data reveal the crucial role of NEK8 kinase activation within the IC, promoting proper left–right patterning, cardiopulmonary development and renal morphogenesis.

Tubulin genes encode a series of homologous proteins used to construct microtubules which are essential for multiple cellular processes. Neural development is particularly reliant on functional microtubule structures. Tubulin genes comprise a large family of genes with very high sequence similarity between multiple family members. Human genetics has demonstrated that a large spectrum of cortical malformations are associated with de novo heterozygous mutations in tubulin genes. However, the absolute requirement for many of these genes in development and disease has not been previously tested in genetic loss of function models. Here we directly test the requirement for Tuba1a, Tubb2a and Tubb2b in the mouse by deleting each gene individually using CRISPR-Cas9 genome editing. We show that loss of Tubb2a or Tubb2b does not impair survival but does lead to relatively mild cortical malformation phenotypes. In contrast, loss of Tuba1a is perinatal lethal and leads to significant forebrain dysmorphology. We also present a novel mouse ENU allele of Tuba1a with phenotypes similar to the null allele. This demonstrates the requirements for each of the tubulin genes and levels of functional redundancy are quite different throughout the gene family. The ability of the mouse to survive in the absence of some tubulin genes known to cause disease in humans suggests future intervention strategies for these devastating tubulinopathy diseases. Tubulin proteins are assembled into microtubules to provide essential cellular cytoskeletal elements. Microtubules are especially crucial for neuronal development. Multiple studies demonstrate that human malformations of cortical development are caused by genetic variants in several tubulin genes. Interestingly, most of these developmental phenotypes are the result of de novo, heterozygous variants likely to act as dominant negatives interfering with normal tubulin function(s). The roles of individual tubulin genes and the mechanism(s) leading to these malformations remain unclear. Genome sequencing efforts revealed high sequence similarity between many tubulin genes, raising the possibility of functional compensation. The requirements for many of these tubulin genes have not been previously addressed in loss of function experiments. We have generated novel deletions of several tubulin genes known to cause human disease to assess if they are required for brain development, or if the human variants act to alter function of the tubulin proteins leading to the pathogenesis. Surprisingly, our results show that some individual tubulin genes are absolutely required for survival while others are not and have much more benign cortical malformations.

The congenital polycystic kidney (cpk) mutation is the most extensively characterized mouse model of polycystic kidney disease (PKD). The renal cystic disease is fully expressed in homozygotes and is strikingly similar to human autosomal recessive PKD (ARPKD), whereas genetic background modulates the penetrance of the corresponding defect in the developing biliary tree. We now describe the positional cloning, mutation analysis, and expression of a novel gene that is disrupted in cpk mice. The cpk gene is expressed primarily in the kidney and liver and encodes a hydrophilic, 145–amino acid protein, which we term cystin. When expressed exogenously in polarized renal epithelial cells, cystin is detected in cilia, and its expression overlaps with polaris, another PKD-related protein. We therefore propose that the single epithelial cilium is important in the functional differentiation of polarized epithelia and that ciliary dysfunction underlies the PKD phenotype in cpk mice.

We have previously described a forward genetic screen in mice for abnormalities of brain development. Characterization of two hydrocephalus mutants by whole-exome sequencing after whole-genome SNP mapping revealed novel recessive mutations in Dnaaf1 and Lrrc48. Mouse mutants of these two genes have not been previously reported. The Dnaaf1 mutant carries a mutation at the splice donor site of exon 4, which results in abnormal transcripts. The Lrrc48 mutation is a missense mutation at a highly conserved leucine residue, which is also associated with a decrease in Lrrc48 transcription. Both Dnaaf1 and Lrrc48 belong to a leucine-rich repeat-containing protein family and are components of the ciliary axoneme. Their Chlamydomonas orthologs are known to be required for normal ciliary beat frequency or flagellar waveform, respectively. Some Dnaaf1 or Lrrc48 homozygote mutants displayed laterality defects, suggesting a motile cilia defect in the embryonic node. Mucus accumulation and neutrophil infiltration in the maxillary sinuses suggested sinusitis. Dnaaf1 mutants showed postnatal lethality, and none survived to weaning age. Lrrc48 mutants survive to adulthood, but had male infertility. ARL13B immunostaining showed the presence of motile cilia in the mutants, and the distal distribution of DNAH9 in the axoneme of upper airway motile cilia appeared normal. The phenotypic abnormalities suggest that mutations in Dnaaf1 and Lrrc48 cause defects in motile cilia function.