NI-024 Reveals Primary Microglial Deficits in CRL

(Invent Biotechnologies Inc.)

CSF1R-related leukoencephalopathy (CRL) is a devastating adult-onset neurodegenerative disorder caused by dominant mutations in the colony-stimulating factor-1 receptor (CSF1R). The disease is characterized by progressive cognitive decline, motor dysfunction, psychiatric abnormalities, white matter degeneration, and premature death. Although previous studies established that CRL is fundamentally a microglial disease, the primary cellular defect that initiates pathology remained unknown.

In a landmark study, Chițu et al. (2026) employed single-nucleus RNA sequencing (snRNA-seq), metallomic analyses, proteomics, and behavioral studies to identify the earliest pathogenic events occurring in presymptomatic Csf1r+/– mice. Their findings revealed that impaired CSF1R signaling disrupts metal ion homeostasis in microglia, leading to oxidative stress, altered communication with oligodendrocytes and neurons, and ultimately neurodegeneration. A key enabling technology for this work was the use of the Invent Biotechnologies Detergent-Free Nuclear Isolation Kit (NI-024), which provided high-quality nuclei suitable for comprehensive single-nucleus transcriptomic analysis.

NI-024 Enables High-Quality Single-Nucleus Profiling of the Diseased Brain

The central objective of the study was to investigate molecular changes occurring before the appearance of pathological lesions or behavioral symptoms. To achieve this, the authors performed snRNA-seq on brains from 2-month-old Csf1r+/– mice and wild-type controls.

Isolation of intact nuclei from brain tissue represents a major technical challenge because conventional detergent-based approaches, in some cases, could damage nuclear membranes, alter transcript representation, increase ambient RNA contamination, and compromise downstream sequencing quality. To overcome these limitations, the investigators utilized the Invent Biotechnologies detergent-free nuclei isolation kit.

The quality of the resulting dataset demonstrates the effectiveness of the NI-024 workflow. The study successfully generated and analyzed 75,141 individual nuclei from six mouse brains, yielding excellent transcript recovery and allowing identification of 31 distinct cellular clusters representing: Microglia, Oligodendrocytes, Endothelial cells, Astrocytes, Multiple excitatory neuronal populations and Multiple inhibitory neuronal populations.

Figure 1. snRNA-seq identifies major populations of neurons and glial cells and shows selective reduction of neuronal subpopulations in Csf1r+/– mice. (A) Diagram of the snRNA-seq workflow. (B) Uniform manifold approximation and projection (UMAP) plot showing the clustering of nuclei. n = 3 mouse brain samples per genotype; 75,141 total nuclei. (C) Dot plot showing the expression of cell–type–specific markers. (D) Dot plot showing the expression of neuronal markers of cortical layers and callosal projection neurons (CPN). (E) Pie chart showing the frequency of each cluster. (F) Relative frequencies of WT and Csf1r+/– nuclei in each cluster. No statistically significant change was detected. Two-way ANOVA, Bonferroni. (G) Relative frequency of clusters in WT and Csf1r+/– brains normalized to the overall frequency in E. (H) Number of differentially expressed genes (DEGs) in each cluster. The data were obtained from 3 mice/genotype. UR, upregulated; DR downregulated in Csf1r+/–.

The ability to obtain such a large and diverse population of intact nuclei was essential for detecting subtle transcriptomic alterations occurring during the earliest stages of disease development.

Discovery of the Primary Microglial Defect

The snRNA-seq dataset generated using NI-024 enabled the investigators to determine which cell populations were most affected by CSF1R haploinsufficiency.

Analysis revealed that microglia exhibited the largest number of differentially expressed genes, confirming that microglia is the primary cellular target of CSF1R deficiency. Importantly, the study examined animals before overt pathology developed, allowing the observed changes to be interpreted as primary disease mechanisms rather than secondary responses to neurodegeneration. Transcriptomic analysis demonstrated that Csf1r+/– microglia displayed altered expression of genes involved in: Metal ion transport, Mitochondrial metabolism, Inflammatory signaling and Oxidative stress regulation.

Significance of NI-024 for Neurodegenerative Disease Research

The Invent Biotechnologies NI-024 kit played a foundational role in this study by enabling the generation of high-quality nuclei suitable for large-scale single-nucleus transcriptomic analysis. The detergent-free isolation approach preserved nuclear integrity and transcript representation, allowing the investigators to identify disease-associated molecular signatures that would likely be obscured in bulk tissue analyses.

Conclusion

Chițu and colleagues provide compelling evidence that disruption of metal ion homeostasis is a primary consequence of reduced CSF1R signaling and a major driver of CRL pathogenesis. Their work identifies metallothionein-regulated zinc, copper, and iron metabolism as critical determinants of microglial health and neurodegeneration.

The success of this investigation depended heavily on the use of the Invent Biotechnologies NI-024 Detergent-Free Nuclear Isolation Kit, which enabled high-quality single-nucleus RNA sequencing from adult mouse brain tissue. By preserving nuclear integrity and supporting robust transcriptomic profiling, NI-024 facilitated the discovery of previously unrecognized mechanisms linking microglial dysfunction to progressive white matter disease.

Reference

Chițu V, Alvarenga J, Chen W, Reynolds D, Liu Y, Sun D, et al. Disruption of CSF-1 receptor-mediated metal ion homeostasis in the murine brain promotes neurodegenerative disease. Journal of Clinical Investigation. 2026;136(9). DOI: 10.1172/JCI200121.