Neural stem/progenitor cells (NSPCs) are the stem cells that give rise to the entire brain and even continue to form new neurons throughout life. Understanding what regulates NSPC behavior is thus important for development and adulthood. Recently, metabolism has been shown to have an important raie in the regulation of stem cell activity/fate in different tissues. Previously, Knobloch and colleagues described the importance of lipid metabolism for murine NSPCs, specifically the build-up of lipids by de novo lipogenesis and the break-down of lipids via fatty acid oxidation (FAO). They showed that these two pathways were implicated in regulating NSPC quiescence, proliferation and integration of their progeny in the mouse brain. ln a follow up study from our group, Ramosaj, Madsen and colleagues showed that the lipid storing organelles called lipid droplets (LDs) also play an important raie for NSPC metabolism and proliferation.
However, whether lipid metabolism or cellular lipid mobilization play a similar role in the regulation of human NSPCs (hNSPCs) remains poorly understood. Recently, Gonzalez-Bohorquez and colleagues showed that blocking de novo lipogenesis in NSPCs interfered with NSPC function, causing a disorganization of the cellular structure of the developing mouse brain, and affecting hNSCPs. Whether FAO and LDs play an equally important role for hNSPC remains unknown.
To study the raie of FAO and LDs in a human context, we established a series of in vitro models to mimic different aspects of hNSPC function and human brain development, ranging from proliferative hNSPCs, quiescent hNSPCs, differentiated hNSPC, neural rosette formation, cerebral organoids and forebrain organoids.
Using this combination of models, we first targeted carnitine palmitoyl transferase 1A (CPT1A), the rate­ limiting enzyme of FAO, using the pharmacological FAO inhibitor etomoxir and shRNAs against CPT1A. Our results show that CPT1A is highly expressed in hNSPCs during brain development. While blocking FAO in hNSPC monocultures only showed subtle effects on proliferation, blocking FAO in cerebral organoids strongly reduced hNSPC proliferation. Blocking FAO also increased cell death bath in hNSPCs and cerebral organoids, suggesting that FAO is indeed an important metabolic pathway for hNSPCs.
ln addition, we used the in vitro models to study the effects of a novel double point mutation of a patient presenting severe cognitive impairment and metabolic decompensations. Sequencing studies showed that the patient had a de novo compound heterozygous mutation in PUNS, a LD-coat protein which promotes the association of LDs with mitochondria. Given the important raie of LDs in mouse NSPCs, we were interested in studying whether these mutations might affect hNSPC behavior. The patient cell lines had difficulties inducing towards the neural lineage, and the cells that managed to turn into hNSPCs had a higher tendency to differentiate. Furthermore, preliminary data suggests that the patient's NSPCs might shift towards using more FAO. Taken together, the data suggests that mutations in PLINS indeed seem to affect the patient's NSPCs.
The research conducted in this thesis suggests that hNSPC lipid metabolism and lipid mobilization via LDs play an important role in brain development by both regulating induction towards neural lineage as well as NSPC behavior.