Novel roles for the ectoenzyme CD38 in the maintenance of transcriptional and metabolic homeostasis in astrocytes

CD38 is an ectoenzyme that converts NAD+ to NAM and helps to maintain bioenergetic homeostasis. A better understanding of CD38 biology in the brain is needed, considering the evidence for CD38 dysregulation and/or gene variation in neurodegenerative conditions such as Parkinson's Disease (PD) and Alzheimer's Disease (AD). Here, we demonstrate enrichment of Cd38 in midbrain astrocytes and describe how CD38 deficiency influences brain metabolism, astrocytic gene expression, and bioenergetics. We demonstrate increased NAD content, decreased NAM content, and increased NAD/NAM in the midbrain and striatum of CD38-deficient (Cd38-/-) mice, indicating the dependence on CD38 for NAD to NAM conversion. RNA-sequencing of isolated astrocytes revealed numerous differentially expressed genes in Cd38+/- and Cd38-/- mice, with alterations in mitochondrial, metabolic, and senescence-related genes and several genes involved in PD and AD etiology. Interestingly, Seahorse XFe24 mitochondrial functional evaluation in primary astrocyte cultures from Cd38+/+, Cd38+/-, and Cd38-/- mice revealed enhanced oxygen consumption, with an increase in glycolytic rate with Cd38 deficiency. These findings indicate a novel role for astrocytes in the regulation of CD38-dependent NAD/NAM homeostasis in the brain and provide the framework for future studies evaluating the relationship between CD38 dysfunction, aging, and vulnerability of neuronal populations in neurodegenerative disease. Also, these studies highlight the need to better elucidate the impact of CD38 deficiency on brain metabolism, considering ongoing clinical trials using CD38 inhibitors for the treatment of multiple myeloma and other cancers. Overall design: Astrocytes were isolated from Cd38+/+, Cd38+/-, and Cd38-/- late (n = 3, 3 and 6 respectively) -adulthood male mice after 1 year of age (12-14 months, x¯ = 13.5 months) utilizing a magnetic isolation bead approach (MACS -Miltenyi). Midbrain regions associated with Parkinson's disease pathology (striatum, thalamus, and substantia nigra) were microdissected from brain tissue for cell-specific isolation. Tissue dissociation was performed, whereby regions of interest were utilized for MACS. All dissections were performed in modified artificial cerebral spinal fluid (120 mM NaCl, 3 mM KCl, 6 mM 2NaHCO3, 11 mM glucose, 5 mM HEPES, 200 µM CaCl, 1 mM MgCl) containing neuronal activity inhibitors (20 µM CNQX, 20 µM D-AP5, 295-299 mOsm) to reduce glutamate excitotoxicity. Briefly, midbrain tissues were removed then sectioned into 1mm pieces, followed by papain dissociation (Worthington Biochemical Corporation, Catalog# LK003150). A series of centrifugation (4°C, 300 RCF, 3-5 minutes, Eppendorf Catalog# 58048R) and PBS wash steps were performed to retain cell containing tissue pellets. Supernatants were discarded and pellets were resuspended in 0.5% w/v bovine serum albumin (Millipore Sigma, Catalog# A7030) in PBS for filtration (70 µm, Fisher Scientific Catalog# 22-363-548) and attainment of midbrain cellular fractions. Cell fractions were then treated with Miltenyi Biotec Microbeads to remove contaminating endothelial cells (Catalog# 130-097-418) and myelin/oligodendrocytes (Catalog# 130-096-433). Samples were incubated in microbeads for 10 minutes at 4°C followed by filtering through Miltenyi LS columns (Catalog# 130-042-401). Samples were then treated with anti-CD11b microbeads (Catalog# 130-092-263) for 10 minutes at 4°C to selectively isolate microglia by elution of magnetically captured cells for collection. Finally, astrocytes were positively selected using the Miltenyi ACSA-II microbead kit (Catalog# 130-097-679). Samples were first incubated with an FcR blocking reagent for 10 minutes at 4°C immediately followed by an additional 10-minute incubation at 4°C with anti-ACSA-2 microbeads. After LS column filtration and elution, collected cells were stored in 300 µL Trizol (ThermoFisher Scientific, Catalog# 15596026) and snap frozen before being placed in storage at -80°C. Astrocytes were removed from -80°C , placed on ice to thaw and then gently lysed by syringe for preparation of RNA isolation (Zymo Research, Catalog# R2062). RNA was extracted according to the Direct-zol RNA Microprep protocols, including DNA-degradation with DNase I. Total RNA was eluted in 10µL of DNAse/RNAse free water. Samples were submitted to Medgenome for rRNA depletion, quality verifications, library construction, and ultra-low input RNA-sequencing. RNA integrity was determined by Qubit fluorometric quantitation and tapestation bioanalyzer analysis. Collected RNA was then converted to cDNA samples by using the Takara SMART-Seq v4Ultra low Input RNA kit. Generated cDNA was checked for quality utilizing Quibit and tapestation measures, whereby libraries with marginal pass and higher quality for were used for pair-ended sequencing on the NovaSeq 6000 platform.