Distinct disease mutations in DNMT3A result in a spectrum of behavioral, epigenetic, and transcriptional deficits.

Phenotypic heterogeneity in monogenic neurodevelopmental disorders can arise from differential severity of variants underlying disease, but how distinct alleles drive variable disease presentation is not well understood. Here, we investigate missense mutations in DNA methyltransferase 3A (DNMT3A), a DNA methyltransferase associated with overgrowth, intellectual disability, and autism, to uncover molecular correlates of phenotypic heterogeneity. We generate a Dnmt3aP900L/+ mouse mimicking a mutation with mild to moderate severity and compare phenotypic and epigenomic effects with a severe R878H mutation. P900L mutants exhibit core growth and behavioral phenotypes shared across models but show subtle epigenomic changes, while R878H mutants display extensive disruptions. We identify mutation-specific dysregulated genes that may contribute to variable disease severity. Shared transcriptomic disruption identified across mutations overlaps dysregulation observed in other developmental disorder models and likely drives common phenotypes. Together, our findings define central drivers of DNMT3A disorders and illustrate how variable epigenomic disruption contributes to phenotypic heterogeneity in neurodevelopmental disease.

Single-Nucleus Transcriptional Profiling of GAD2-Positive Neurons From Mouse Lateral Habenula Reveals Distinct Expression of Neurotransmission- and Depression-Related Genes.

Background: Glutamatergic projection neurons of the lateral habenula (LHb) drive behavioral state modulation by regulating the activity of midbrain monoaminergic neurons. Identifying circuit mechanisms that modulate LHb output is of interest for understanding control of motivated behaviors. Methods: A small population of neurons within the medial subnucleus of the mouse LHb express the GABAergic (gamma-aminobutyric acidergic)-synthesizing enzyme GAD2, and they can inhibit nearby LHb projection neurons; however, these neurons lack markers of classic inhibitory interneurons, and they coexpress the vesicular glutamate transporter VGLUT2. To determine the molecular phenotype of these neurons, we genetically tagged the nuclei of GAD2-positive cells and used fluorescence-activated nuclear sorting to isolate and enrich these nuclei for single-nucleus RNA sequencing. Results: Our data confirm that GAD2+/VGLUT2+ neurons intrinsic to the LHb coexpress markers of both glutamatergic and GABAergic transmission and that they are transcriptionally distinct from either GABAergic interneurons or habenular glutamatergic neurons. We identify gene expression programs within these cells that show sex-specific differences in expression and that are implicated in major depressive disorder, which has been linked to LHb hyperactivity. Finally, we identify the Ntng2 gene encoding the cell adhesion protein netrin-G2 as a marker of LHb GAD2+/VGLUT2+ neurons and a gene product that may contribute to their target projections. Conclusions: These data show the value of using genetic enrichment of rare cell types for transcriptome studies, and they advance understanding of the molecular composition of a functionally important class of GAD2+ neurons in the LHb.

Pharmacodynamics of ATI-2307 in a rabbit model of cryptococcal meningoencephalitis.

Cryptococcal meningoencephalitis (CM) is a devastating fungal disease with high morbidity and mortality. The current regimen that is standard-of-care involves a combination of three different drugs administered for up to one year. There is a critical need for new therapies due to both toxicity and inadequate fungicidal activity of the currently available antifungal drugs. ATI-2307 is a novel aryl amidine that disrupts the mitochondrial membrane potential and inhibits the respiratory chain complexes of fungi-it thus represents a new mechanism for direct antifungal action. Furthermore, ATI-2307 selectively targets fungal mitochondria via a fungal-specific transporter that is not present in mammalian cells. It has very potent in vitro anticryptococcal activity. In this study, the efficacy of ATI-2307 was tested in a rabbit model of CM. ATI-2307 demonstrated significant fungicidal activity at dosages between 1 and 2 mg/kg/d, and these results were superior to fluconazole and similar to amphotericin B treatment. When ATI-2307 was combined with fluconazole, the antifungal effect was greater than either therapy alone. While ATI-2307 has potent anticryptococcal activity in the subarachnoid space, its ability to reduce yeasts in the brain parenchyma was relatively less over the same study period. This new drug, with its unique mechanism of fungicidal action and ability to positively interact with an azole, has demonstrated sufficient anticryptococcal potential in this experimental setting to be further evaluated in clinical studies.

Single-nucleus transcriptional profiling of GAD2-positive neurons from mouse lateral habenula reveals distinct expression of neurotransmission- and depression-related genes.

Glutamatergic projection neurons of the lateral habenula (LHb) drive behavioral state modulation by regulating the activity of midbrain monoaminergic neurons. Identifying circuit mechanisms that modulate LHb output is of interest for understanding control of motivated behaviors. A small population of neurons within the medial subnucleus of the mouse LHb express the GABAergic synthesizing enzyme GAD2, and they can inhibit nearby LHb projection neurons; however, these neurons lack markers of classic inhibitory interneurons and they co-express the vesicular glutamate transporter VGLUT2. To determine the molecular phenotype of these neurons, we genetically tagged the nuclei of GAD2-positive cells and used fluorescence-activated nuclear sorting to isolate and enrich these nuclei for single nuclear RNA sequencing (FANS-snRNAseq). Our data confirm that GAD2+/VGLUT2+ neurons intrinsic to the LHb co-express markers of both glutamatergic and GABAergic transmission and that they are transcriptionally distinct from either GABAergic interneurons or habenular glutamatergic neurons. We identify gene expression programs within these cells that show sex-specific differences in expression and that are implicated in major depressive disorder (MDD), which has been linked to LHb hyperactivity. Finally, we identify the Ntng2 gene encoding the cell adhesion protein Netrin-G2 as a marker of LHb GAD2+/VGLUT+ neurons and a gene product that may contribute to their target projections. These data show the value of using genetic enrichment of rare cell types for transcriptome studies, and they advance understanding of the molecular composition of a functionally important class of GAD2+ neurons in the LHb.

Efficacy of APX2039 in a Rabbit Model of Cryptococcal Meningitis.

Cryptococcal Meningitis (CM) is uniformly fatal if not treated, and treatment options are limited. We previously reported on the activity of APX2096, the prodrug of the novel Gwt1 inhibitor APX2039, in a mouse model of CM. Here, we investigated the efficacy of APX2039 in mouse and rabbit models of CM. In the mouse model, the controls had a mean lung fungal burden of 5.95 log10 CFU/g, whereas those in the fluconazole-, amphotericin B-, and APX2039-treated mice were 3.56, 4.59, and 1.50 log10 CFU/g, respectively. In the brain, the control mean fungal burden was 7.97 log10 CFU/g, while the burdens were 4.64, 7.16, and 1.44 log10 CFU/g for treatment with fluconazole, amphotericin B, and APX2039, respectively. In the rabbit model of CM, the oral administration of APX2039 at 50 mg/kg of body weight twice a day (BID) resulted in a rapid decrease in the cerebrospinal fluid (CSF) fungal burden, and the burden was below the limit of detection by day 10 postinfection. The effective fungicidal activity (EFA) was -0.66 log10 CFU/mL/day, decreasing from an average of 4.75 log10 CFU/mL to 0 CFU/mL, over 8 days of therapy, comparing favorably with good clinical outcomes in humans associated with reductions of the CSF fungal burden of -0.4 log10 CFU/mL/day, and, remarkably, 2-fold the EFA of amphotericin B deoxycholate in this model (-0.33 log10 CFU/mL/day). A total drug exposure of the area under the concentration-time curve from 0 to 24 h (AUC0-24) of 25 to 50 mg · h/L of APX2039 resulted in near-maximal antifungal activity. These data support the further preclinical and clinical evaluation of APX2039 as a new oral fungicidal monotherapy for the treatment of CM. IMPORTANCE Cryptococcal meningitis (CM) is a fungal disease with significant global morbidity and mortality. The gepix Gwt1 inhibitors are a new class of antifungal drugs. Here, we demonstrated the efficacy of APX2039, the second member of the gepix class, in rabbit and mouse models of cryptococcal meningitis. We also analyzed the drug levels in the blood and cerebrospinal fluid in the highly predictive rabbit model and built a mathematical model to describe the behavior of the drug with respect to the elimination of the fungal pathogen. We demonstrated that the oral administration of APX2039 resulted in a rapid decrease in the CSF fungal burden, with an effective fungicidal activity of -0.66 log10 CFU/mL/day, comparing favorably with good clinical outcomes in humans associated with reductions of -0.4 log10 CFU/mL/day. The drug APX2039 had good penetration of the central nervous system and is an excellent candidate for future clinical testing in humans for the treatment of CM.

Filtered Cerebrospinal Fluid From Patients With Amyotrophic Lateral Sclerosis Displays an Altered Proteome and Affects Motor Phenotype in a Mouse Model.

INTRODUCTION: Cerebrospinal fluid (CSF) has been implicated in amyotrophic lateral sclerosis (ALS) due to its ability to spread inflammatory proteins throughout the nervous system. We hypothesized that filtration of the CSF could remove pathogenic proteins and prevent them from altering motor phenotypes in a mouse model. METHODS: We filtered the CSF from 11 ALS patients via 100 kilodaltons (kD) molecular weight cut-off filters. We used mass spectrometry-based discovery proteomics workflows to compare protein abundances before and after filtration. To test the effects of CSF filtration on motor function, we injected groups of mice with saline, filtered ALS-CSF, or unfiltered ALS-CSF (n=12 per group) and assessed motor function via pole descent and open field tests. RESULTS: We identified proteins implicated in ALS pathogenesis and showed that these were removed in significant amounts in our workflow. Key filtered proteins included complement proteins, chitinases, serine protease inhibitors, and neuro-inflammatory proteins such as amyloid precursor protein, chromogranin A, and glial fibrillary acidic protein. Compared to the filtered ALS-CSF mice, unfiltered ALS-CSF mice took longer to descend a pole (10 days post-injection, 11.14 seconds vs 14.25 seconds, p = 0.02) and explored less on an open field (one day post-injection, 21.81 m vs 16.83 m, p = 0.0004). CONCLUSIONS: We demonstrated the ability to filter proteins from the CSF of ALS patients and identified potentially pathologic proteins that were reduced in quantity. Additionally, we demonstrated the ability of unfiltered ALS-CSF to induce motor deficits in mice on the pole descent and open field tests and showed that filtration could prevent this deficit. Given the lack of effective treatments for ALS, this could be a novel solution for patients suffering from this deadly and irreversible condition.

Evaluation of neurapheresis therapy in vitro: a novel approach for the treatment of leptomeningeal metastases.

BACKGROUND: Leptomeningeal metastases (LM), late-stage cancer when malignant cells migrate to the subarachnoid space (SAS), have an extremely poor prognosis. Current treatment regimens fall short in effectively reducing SAS tumor burden. Neurapheresis therapy is a novel approach employing filtration and enhanced circulation of the cerebrospinal fluid (CSF). Here, we examine the in vitro use of neurapheresis therapy as a novel, adjunctive treatment option for LM by filtering cells and augmenting the distribution of drugs that may have the potential to enhance the current clinical approach. METHODS: Clinically relevant concentrations of VX2 carcinoma cells were suspended in artificial CSF. The neurapheresis system's ability to clear VX2 carcinoma cells was tested with and without the chemotherapeutic presence (methotrexate [MTX]). The VX2 cell concentration following each filtration cycle and the number of cycles required to reach the limit of detection were calculated. The ability of neurapheresis therapy to circulate, distribute, and maintain therapeutic levels of MTX was assessed using a cranial-spinal model of the SAS. The distribution of a 6 mg dose was monitored for 48 h. An MTX-specific ELISA measured drug concentration at ventricular, cervical, and lumbar sites in the model over time. RESULTS: In vitro filtration of VX2 cancer cells with neurapheresis therapy alone resulted in a 2.3-log reduction in cancer cell concentration in 7.5 h and a 2.4-log reduction in live-cancer cell concentration in 7.5 h when used with MTX. Cranial-spinal model experiments demonstrated the ability of neurapheresis therapy to enhance the circulation of MTX in CSF along the neuraxis. CONCLUSION: Neurapheresis has the potential to act as an adjunct therapy for LM patients and significantly improve the standard of care.

The histone demethylase Kdm6b regulates a mature gene expression program in differentiating cerebellar granule neurons.

The histone H3 lysine 27 (H3K27) demethylase Kdm6b (Jmjd3) can promote cellular differentiation, however its physiological functions in neurons remain to be fully determined. We studied the expression and function of Kdm6b in differentiating granule neurons of the developing postnatal mouse cerebellum. At postnatal day 7, Kdm6b is expressed throughout the layers of the developing cerebellar cortex, but its expression is upregulated in newborn cerebellar granule neurons (CGNs). Atoh1-Cre mediated conditional knockout of Kdm6b in CGN precursors either alone or in combination with Kdm6a did not disturb the gross morphological development of the cerebellum. Furthermore, RNAi-mediated knockdown of Kdm6b in cultured CGN precursors did not alter the induced expression of early neuronal marker genes upon cell cycle exit. By contrast, knockdown of Kdm6b significantly impaired the induction of a mature neuronal gene expression program, which includes gene products required for functional synapse maturation. Loss of Kdm6b also impaired the ability of Brain-Derived Neurotrophic Factor (BDNF) to induce expression of Grin2c and Tiam1 in maturing CGNs. Taken together, these data reveal a previously unknown role for Kdm6b in the postmitotic stages of CGN maturation and suggest that Kdm6b may work, at least in part, by a transcriptional mechanism that promotes gene sensitivity to regulation by BDNF.