Towards a Treatment for Leukodystrophy Using Cell-Based Interception and Precision Medicine.

Cell-based interception and precision medicine is a novel approach aimed at improving healthcare through the early detection and treatment of diseased cells. Here, we describe our recent progress towards developing cell-based interception and precision medicine to detect, understand, and advance the development of novel therapeutic approaches through a single-cell omics and drug screening platform, as part of a multi-laboratory collaborative effort, for a group of neurodegenerative disorders named leukodystrophies. Our strategy aims at the identification of diseased cells as early as possible to intercept progression of the disease prior to severe clinical impairment and irreversible tissue damage.

Biallelic pathogenic variants in POLR3D alter tRNA transcription and cause a hypomyelinating leukodystrophy: A case report.

RNA polymerase III-related leukodystrophy (POLR3-related leukodystrophy) is a rare, genetically determined hypomyelinating disease arising from biallelic pathogenic variants in genes encoding subunits of RNA polymerase III (Pol III). Here, we describe the first reported case of POLR3-related leukodystrophy caused by biallelic pathogenic variants in POLR3D, encoding the RPC4 subunit of Pol III. The individual, a female, demonstrated delays in walking and expressive and receptive language as a child and later cognitively plateaued. Additional neurological features included cerebellar signs (e.g., dysarthria, ataxia, and intention tremor) and dysphagia, while non-neurological features included hypodontia, hypogonadotropic hypogonadism, and dysmorphic facial features. Her MRI was notable for diffuse hypomyelination with myelin preservation of early myelinating structures, characteristic of POLR3-related leukodystrophy. Exome sequencing revealed the biallelic variants in POLR3D, a missense variant (c.541C > T, p.P181S) and an intronic splice site variant (c.656-6G > A, p.?). Functional studies of the patient's fibroblasts demonstrated significantly decreased RNA-level expression of POLR3D, along with reduced expression of other Pol III subunit genes. Notably, Pol III transcription was also shown to be aberrant, with a significant decrease in 7SK RNA and several distinct tRNA genes analyzed. Affinity purification coupled to mass spectrometry of the POLR3D p.P181S variant showed normal assembly of Pol III subunits yet altered interaction of Pol III with the PAQosome chaperone complex, indicating the missense variant is likely to alter complex maturation. This work identifies biallelic pathogenic variants in POLR3D as a novel genetic cause of POLR3-related leukodystrophy, expanding the molecular spectrum associated with this disease, and proposes altered tRNA homeostasis as a factor in the underlying biology of this hypomyelinating disorder.

Hypomyelination, hypodontia and craniofacial abnormalities in a Polr3b mouse model of leukodystrophy.

RNA polymerase III (Pol III)-related hypomyelinating leukodystrophy (POLR3-HLD), also known as 4H leukodystrophy, is a severe neurodegenerative disease characterized by the cardinal features of hypomyelination, hypodontia and hypogonadotropic hypogonadism. POLR3-HLD is caused by biallelic pathogenic variants in genes encoding Pol III subunits. While approximately half of all patients carry mutations in POLR3B encoding the RNA polymerase III subunit B, there is no in vivo model of leukodystrophy based on mutation of this Pol III subunit. Here, we determined the impact of POLR3BΔ10 (Δ10) on Pol III in human cells and developed and characterized an inducible/conditional mouse model of leukodystrophy using the orthologous Δ10 mutation in mice. The molecular mechanism of Pol III dysfunction was determined in human cells by affinity purification-mass spectrometry and western blot. Postnatal induction with tamoxifen induced expression of the orthologous Δ10 hypomorph in triple transgenic Pdgfrα-Cre/ERT; R26-Stopfl-EYFP; Polr3bfl mice. CNS and non-CNS features were characterized using a variety of techniques including microCT, ex vivo MRI, immunofluorescence, immunohistochemistry, spectral confocal reflectance microscopy and western blot. Lineage tracing and time series analysis of oligodendrocyte subpopulation dynamics based on co-labelling with lineage-specific and/or proliferation markers were performed. Proteomics suggested that Δ10 causes a Pol III assembly defect, while western blots demonstrated reduced POLR3BΔ10 expression in the cytoplasm and nucleus in human cells. In mice, postnatal Pdgfrα-dependent expression of the orthologous murine mutant protein resulted in recessive phenotypes including severe hypomyelination leading to ataxia, tremor, seizures and limited survival, as well as hypodontia and craniofacial abnormalities. Hypomyelination was confirmed and characterized using classic methods to quantify myelin components such as myelin basic protein and lipids, results which agreed with those produced using modern methods to quantify myelin based on the physical properties of myelin membranes. Lineage tracing uncovered the underlying mechanism for the hypomyelinating phenotype: defective oligodendrocyte precursor proliferation and differentiation resulted in a failure to produce an adequate number of mature oligodendrocytes during postnatal myelinogenesis. In summary, we characterized the Polr3bΔ10 mutation and developed an animal model that recapitulates features of POLR3-HLD caused by POLR3B mutations, shedding light on disease pathogenesis, and opening the door to the development of therapeutic interventions.

A Recurrent De Novo Variant in EIF2AK2 Causes a Hypomyelinating Leukodystrophy.

De novo pathogenic variants in EIF2AK2 have recently been reported as a novel genetic cause of leukoencephalopathy. Here, we describe a male individual who presented in the first year of life with clinical features resembling Pelizaeus-Merzbacher disease (PMD), including nystagmus, hypotonia, and global developmental delay, and which later progressed to include ataxia and spasticity. Brain MRI at the age of two revealed diffuse hypomyelination. This report adds to the limited number of individuals published and further reinforces de novo variants in EIF2AK2 as a molecular cause of a leukodystrophy that clinically and radiologically resembles PMD.

Decreased RNA polymerase III subunit expression leads to defects in oligodendrocyte development.

Introduction: RNA polymerase III (Pol III) is a critical enzymatic complex tasked with the transcription of ubiquitous non-coding RNAs including 5S rRNA and all tRNA genes. Despite the constitutive nature of this enzyme, hypomorphic biallelic pathogenic variants in genes encoding subunits of Pol III lead to tissue-specific features and cause a hypomyelinating leukodystrophy, characterized by a severe and permanent deficit in myelin. The pathophysiological mechanisms in POLR3- related leukodystrophy and specifically, how reduced Pol III function impacts oligodendrocyte development to account for the devastating hypomyelination seen in the disease, remain poorly understood. Methods: In this study, we characterize how reducing endogenous transcript levels of leukodystrophy-associated Pol III subunits affects oligodendrocyte maturation at the level of their migration, proliferation, differentiation, and myelination. Results: Our results show that decreasing Pol III expression altered the proliferation rate of oligodendrocyte precursor cells but had no impact on migration. Additionally, reducing Pol III activity impaired the differentiation of these precursor cells into mature oligodendrocytes, evident at both the level of OL-lineage marker expression and on morphological assessment, with Pol III knockdown cells displaying a drastically more immature branching complexity. Myelination was hindered in the Pol III knockdown cells, as determined in both organotypic shiverer slice cultures and co-cultures with nanofibers. Analysis of Pol III transcriptional activity revealed a decrease in the expression of distinct tRNAs, which was significant in the siPolr3a condition. Discussion: In turn, our findings provide insight into the role of Pol III in oligodendrocyte development and shed light on the pathophysiological mechanisms of hypomyelination in POLR3-related leukodystrophy.

An optimized and validated protocol for the purification of PDGFRα+ oligodendrocyte precursor cells from mouse brain tissue via immunopanning.

Immunopanning is an efficient and reliable method for isolating primary cells from rodent brain tissue, making it a valuable tool for researchers interested in in vitro glial models. Here, we present an immunopanning protocol optimized for the isolation of Platelet-Derived Growth Factor Receptor Alpha positive (PDGFRα+) oligodendrocyte precursor cells (OPCs) from mouse brain tissue that results in a high yield of pure OPCs from minimal quantities of starting tissue.•The protocol presented here is optimized for a PDGFRα-dependent selection of mouse OPCs using a commercial antibody, accounting for the relatively weaker adhesion of OPCs to the anti-PDGFRα plate as compared to other oligodendrocyte lineage markers (e.g., MOG).•A modified papain digestion step, with 95% O2/5% CO2 gas that is humidified prior to perfusion, significantly enhances the yield of dissociated cells and final yield of OPCs.•Isolating OPCs at the PDGFRα+ stage permits the expansion of cells in culture, facilitating studies using transgenic mice, and enables studies on the development of the oligodendrocyte lineage without the spatial and temporal complexity of in vivo studies.

RNA Polymerases I and III in development and disease.

Ribosomes are macromolecular machines that are globally required for the translation of all proteins in all cells. Ribosome biogenesis, which is essential for cell growth, proliferation and survival, commences with transcription of a variety of RNAs by RNA Polymerases I and III. RNA Polymerase I (Pol I) transcribes ribosomal RNA (rRNA), while RNA Polymerase III (Pol III) transcribes 5S ribosomal RNA and transfer RNAs (tRNA) in addition to a wide variety of small non-coding RNAs. Interestingly, despite their global importance, disruptions in Pol I and Pol III function result in tissue-specific developmental disorders, with craniofacial anomalies and leukodystrophy/neurodegenerative disease being among the most prevalent. Furthermore, pathogenic variants in genes encoding subunits shared between Pol I and Pol III give rise to distinct syndromes depending on whether Pol I or Pol III function is disrupted. In this review, we discuss the global roles of Pol I and III transcription, the consequences of disruptions in Pol I and III transcription, disorders arising from pathogenic variants in Pol I and Pol III subunits, and mechanisms underpinning their tissue-specific phenotypes.

Novel biallelic variants in NRROS associated with a lethal microgliopathy, brain calcifications, and neurodegeneration.

Negative regulator of reactive oxygen species (NRROS) is a leucine-rich repeat protein expressed by microglia and perivascular macrophages. To date, 9 individuals have been reported with biallelic NRROS variants. Here, we report one individual with a severe neurodegenerative phenotype in which exome sequencing identified 2 novel variants in NRROS, a missense variant (c.185T>C, p.Leu62Pro) and a premature stop codon (c.310C>T, p.Gln104Ter). Pathological examination revealed both extensive grey and white matter involvement, dystrophic calcifications, and infiltration of foamy macrophages. This is the first reported case of NRROS variants with a mitochondrial ultrastructure abnormality noted on electron microscopy analysis of post-mortem tissue.