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Cortical pathology in Vanishing white matter

ABSTRACT: Vanishing white matter (VWM) is classified as a leukodystrophy with astrocytes as primary drivers in its pathogenesis. Magnetic resonance imaging has documented the progressive thinning of cortices in long-surviving patients. Routine histopathological analyses, however, have not yet pointed to cortical involvement in VWM. Here, we provide a comprehensive analysis of the VWM cortex. We employed high-resolution-mass-spectrometry-based proteomics and immunohistochemistry to gain insight into possible molecular disease mechanisms in the cortices of VWM patients. The proteome analysis revealed 268 differentially expressed proteins in the VWM cortices compared to the controls. A majority of these proteins formed a major protein interaction network. A subsequent gene ontology analysis identified enrichment for terms such as cellular metabolism, particularly mitochondrial activity. Importantly, some of the proteins with the most prominent changes in expression were found in astrocytes, indicating cortical astrocytic involvement. Indeed, we confirmed that VWM cortical astrocytes exhibit morphological changes and are less complex in structure than control cells. Our findings also suggest that these astrocytes are immature and not reactive. Taken together, we provide insights into cortical involvement in VWM, which has to be taken into account when developing therapeutic strategies.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Brain

DISEASE(S): Leukoencephalopathy With Vanishing White Matter

SUBMITTER: Jodie Man

LAB HEAD: Marianna Bugiani

PROVIDER: PXD030831 | Pride | 2023-01-02

REPOSITORIES: Pride

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			Action	DRS
	20190704_OR16_UM_Man00014_SA_Exp5_FR_GM_CTRL1_1p5ug.raw	Raw
	20190704_OR16_UM_Man00014_SA_Exp5_FR_GM_CTRL2_1p5ug.raw	Raw
	20190704_OR16_UM_Man00014_SA_Exp5_FR_GM_CTRL3_1p5ug.raw	Raw
	20190704_OR16_UM_Man00014_SA_Exp5_FR_GM_CTRL4_1p5ug.raw	Raw
	20190704_OR16_UM_Man00014_SA_Exp5_FR_GM_VWM1_1p5ug.raw	Raw

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Project description:The central nervous system normally functions at O2 levels which would be regarded as hypoxic by most other tissues. However, most in vitro studies of neurons and astrocytes are conducted under hyperoxic conditions without consideration of O2-dependent cellular adaptation. We analyzed the reactivity of astrocytes to 1, 4 and 9% O2 tensions compared to the cell culture standard of 20% O2, to investigate their ability to sense and translate this O2 information to transcriptional activity. Variance of ambient O2 tension for rat astrocytes resulted in profound changes in ribosomal activity, cytoskeletal and energy-regulatory mechanisms and cytokine-related signaling. Clustering of transcriptional regulation patterns revealed four distinct response pattern groups that directionally pivoted around the 4% O2 tension, or demonstrated coherent ascending/decreasing gene expression patterns in response to diverse oxygen tensions. Immune response and cell cycle/cancer-related signaling pathway transcriptomic subsets were significantly activated with increasing hypoxia, whilst hemostatic and cardiovascular signaling mechanisms were attenuated with increasing hypoxia. Our data indicate that variant O2 tensions induce specific and physiologically-focused transcript regulation patterns that may underpin important physiological mechanisms that connect higher neurological activity to astrocytic function and ambient oxygen environments. These strongly defined patterns demonstrate a strong bias for physiological transcript programs to pivot around the 4% O2 tension, while uni-modal programs that do not, appear more related to pathological actions. The functional interaction of these transcriptional ‘programs’ may serve to regulate the dynamic vascular responsivity of the central nervous system during periods of stress or heightened activity. Cerebral cortices were removed from 6-8-day-old Wistar rats, cortical astrocytes were isolated and these cells were maintained in a humidified incubator at 37°C (95% air; 5% CO2). This resulted in a culture of cortical astrocytes, as confirmed by visual inspection the following day and later by glial fibrillary acidic protein immunohistochemistry. Any cortical astrocyte culture that was not homogenous was disposed of and not used in this study. Culture medium was exchanged every 7 days and cells were grown in culture for up to 14 days. 24hr prior to experimentation cells were transferred to an hypoxic workstation equilibrated with either 1%, 4%, 9% or 21% O2 and 5% CO2, with the remaining percentage gas being N2. Once cortical astrocytes had reached approximately 90% confluence (75cm2 flask) they were subjected to hypoxia as above, washed with PBS, removed from the flask base with 0.05% trypsin-EDTA (Gibco) and then gently centrifuged (500xg). The cell pellet was re-suspended in PBS, centrifuged twice more to remove any traces of media, then triturated in 8-10 volumes of RNAlater (Applied Biosystems), frozen and stored at -80oC. Messenger RNA was amplified and labeled with biotin using the standard Illumina protocol (Illumina TotalPrep RNA Amplification Kit Ambion; Austin, TX, cat # IL1791), and hybridized to Illumina's Sentrix Rat Ref-12,v1 Expression BeadChips (Illumina, San Diego, CA). Three replicates from each treatment group (1%, 21%, 4%, and 9% O2) were hybridized and the data was extracted using the Illumina BeadStudio software(v3.4).

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Cortical pathology in Vanishing white matter

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