A role for mutations in AK9 and other genes affecting ependymal cells in idiopathic normal pressure hydrocephalus.

Idiopathic normal pressure hydrocephalus (iNPH) is an enigmatic neurological disorder that develops after age 60 and is characterized by gait difficulty, dementia, and incontinence. Recently, we reported that heterozygous CWH43 deletions may cause iNPH. Here, we identify mutations affecting nine additional genes (AK9, RXFP2, PRKD1, HAVCR1, OTOG, MYO7A, NOTCH1, SPG11, and MYH13) that are statistically enriched among iNPH patients. The encoded proteins are all highly expressed in choroid plexus and ependymal cells, and most have been associated with cilia. Damaging mutations in AK9, which encodes an adenylate kinase, were detected in 9.6% of iNPH patients. Mice homozygous for an iNPH-associated AK9 mutation displayed normal cilia structure and number, but decreased cilia motility and beat frequency, communicating hydrocephalus, and balance impairment. AK9+/- mice displayed normal brain development and behavior until early adulthood, but subsequently developed communicating hydrocephalus. Together, our findings suggest that heterozygous mutations that impair ventricular epithelial function may contribute to iNPH.

Heterozygous FOXJ1 Mutations Cause Incomplete Ependymal Cell Differentiation and Communicating Hydrocephalus.

Heterozygous mutations affecting FOXJ1, a transcription factor governing multiciliated cell development, have been associated with obstructive hydrocephalus in humans. However, factors that disrupt multiciliated ependymal cell function often cause communicating hydrocephalus, raising questions about whether FOXJ1 mutations cause hydrocephalus primarily by blocking cerebrospinal fluid (CSF) flow or by different mechanisms. Here, we show that heterozygous FOXJ1 mutations are also associated with communicating hydrocephalus in humans and cause communicating hydrocephalus in mice. Disruption of one Foxj1 allele in mice leads to incomplete ependymal cell differentiation and communicating hydrocephalus. Mature ependymal cell number and motile cilia number are decreased, and 12% of motile cilia display abnormal axonemes. We observed decreased microtubule attachment to basal bodies, random localization and orientation of basal body patches, loss of planar cell polarity, and a disruption of unidirectional CSF flow. Thus, heterozygous FOXJ1 mutations impair ventricular multiciliated cell differentiation, thereby causing communicating hydrocephalus. CSF flow obstruction may develop secondarily in some patients harboring FOXJ1 mutations. Heterozygous FOXJ1 mutations impair motile cilia structure and basal body alignment, thereby disrupting CSF flow dynamics and causing communicating hydrocephalus.

Evidence for RNA or protein transport from somatic tissues to the male reproductive tract in mouse.

The development of tools to manipulate the mouse genome, including knockout and transgenic technology, has revolutionized our ability to explore gene function in mammals. Moreover, for genes that are expressed in multiple tissues or at multiple stages of development, the use of tissue-specific expression of the Cre recombinase allows gene function to be perturbed in specific cell types and/or at specific times. However, it is well known that putative tissue-specific promoters often drive unanticipated 'off-target' expression. In our efforts to explore the biology of the male reproductive tract, we unexpectedly found that expression of Cre in the central nervous system resulted in recombination in the epididymis, a tissue where sperm mature for ~1-2 weeks following the completion of testicular development. Remarkably, we not only observed reporter expression in the epididymis when Cre expression was driven from neuron-specific transgenes, but also when Cre expression in the brain was induced from an AAV vector carrying a Cre expression construct. A surprisingly wide range of Cre drivers - including six different neuronal promoters as well as the adipose-specific Adipoq Cre promoter - exhibited off-target recombination in the epididymis, with a subset of drivers also exhibiting unexpected activity in other tissues such as the reproductive accessory glands. Using a combination of parabiosis and serum transfer experiments, we find evidence supporting the hypothesis that Cre may be trafficked from its cell of origin to the epididymis through the circulatory system. Together, our findings should motivate caution when interpreting conditional alleles, and suggest the exciting possibility of inter-tissue RNA or protein trafficking in modulation of reproductive biology.

Discovery and Optimization of Tau Targeted Protein Degraders Enabled by Patient Induced Pluripotent Stem Cells-Derived Neuronal Models of Tauopathy.

Accumulation of misfolded, aggregating proteins concurrent with disease onset and progression is a hallmark of neurodegenerative proteinopathies. An important class of these are tauopathies, such as frontotemporal dementia (FTD) and Alzheimer's disease (AD), associated with accumulation of aberrant forms of tau protein in the brain. Pathological tau undergoes abnormal post-translational modifications, misfolding, oligomerization and changes in solubility, cellular redistribution, and spreading. Development and testing of experimental therapeutics that target these pathological tau conformers requires use of cellular models that recapitulate neuronal endogenous, non-heterologous tau expression under genomic and physiological contexts relevant to disease. In this study, we employed FTD-patient induced pluripotent stem cells (iPSC)-derived neurons, expressing a tau variant or mutation, as primary models for driving a medicinal chemistry campaign around tau targeting degrader series. Our screening goal was to establish structure-activity relationships (SAR) for the different chemical series to identify the molecular composition that most efficiently led to tau degradation in human FTD ex vivo neurons. We describe the identification of the lead compound QC-01-175 and follow-up optimization strategies for this molecule. We present three final lead molecules with tau degradation activity in mutant neurons, which establishes potential disease relevance and will drive future studies on specificity and pharmacological properties.

Delivering Therapeutics to Glioblastoma: Overcoming Biological Constraints.

Glioblastoma multiforme is the most lethal intrinsic brain tumor. Even with the existing treatment regimen of surgery, radiation, and chemotherapy, the median survival time is only 15-23 months. The invasive nature of this tumor makes its complete removal very difficult, leading to a high recurrence rate of over 90%. Drug delivery to glioblastoma is challenging because of the molecular and cellular heterogeneity of the tumor, its infiltrative nature, and the blood-brain barrier. Understanding the critical characteristics that restrict drug delivery to the tumor is necessary to develop platforms for the enhanced delivery of effective treatments. In this review, we address the impact of tumor invasion, the molecular and cellular heterogeneity of the tumor, and the blood-brain barrier on the delivery and distribution of drugs using potential therapeutic delivery options such as convection-enhanced delivery, controlled release systems, nanomaterial systems, peptide-based systems, and focused ultrasound.

Novel zebrafish behavioral assay to identify modifiers of the rapid, nongenomic stress response.

When vertebrates face acute stressors, their bodies rapidly undergo a repertoire of physiological and behavioral adaptations, which is termed the stress response. Rapid changes in heart rate and blood glucose levels occur via the interaction of glucocorticoids and their cognate receptors following hypothalamic-pituitary-adrenal axis activation. These physiological changes are observed within minutes of encountering a stressor and the rapid time domain rules out genomic responses that require gene expression changes. Although behavioral changes corresponding to physiological changes are commonly observed, it is not clearly understood to what extent hypothalamic-pituitary-adrenal axis activation dictates adaptive behavior. We hypothesized that rapid locomotor response to acute stressors in zebrafish requires hypothalamic-pituitary-interrenal (HPI) axis activation. In teleost fish, interrenal cells are functionally homologous to the adrenocortical layer. We derived eight frameshift mutants in genes involved in HPI axis function: two mutants in exon 2 of mc2r (adrenocorticotropic hormone receptor), five in exon 2 or 5 of nr3c1 (glucocorticoid receptor [GR]) and two in exon 2 of nr3c2 (mineralocorticoid receptor [MR]). Exposing larval zebrafish to mild environmental stressors, acute changes in salinity or light illumination, results in a rapid locomotor response. We show that this locomotor response requires a functioning HPI axis via the action of mc2r and the canonical GR encoded by nr3c1 gene, but not MR (nr3c2). Our rapid behavioral assay paradigm based on HPI axis biology can be used to screen for genetic and environmental modifiers of the hypothalamic-pituitary-adrenal axis and to investigate the effects of corticosteroids and their cognate receptor interactions on behavior.

Thalidomide promotes degradation of SALL4, a transcription factor implicated in Duane Radial Ray syndrome.

In historical attempts to treat morning sickness, use of the drug thalidomide led to the birth of thousands of children with severe birth defects. Despite their teratogenicity, thalidomide and related IMiD drugs are now a mainstay of cancer treatment; however, the molecular basis underlying the pleiotropic biology and characteristic birth defects remains unknown. Here we show that IMiDs disrupt a broad transcriptional network through induced degradation of several C2H2 zinc finger transcription factors, including SALL4, a member of the spalt-like family of developmental transcription factors. Strikingly, heterozygous loss of function mutations in SALL4 result in a human developmental condition that phenocopies thalidomide-induced birth defects such as absence of thumbs, phocomelia, defects in ear and eye development, and congenital heart disease. We find that thalidomide induces degradation of SALL4 exclusively in humans, primates, and rabbits, but not in rodents or fish, providing a mechanistic link for the species-specific pathogenesis of thalidomide syndrome.