Mitochondrial regulator PGC-1a in neuronal metabolism and brain aging.

The brain is a high energy tissue, and the cell types of which it is comprised are distinct in function and in metabolic requirements. The transcriptional co-activator PGC-1a is a master regulator of mitochondrial function and is highly expressed in the brain; however, its cell-type specific role in regulating metabolism has not been well established. Here, we show that PGC-1a is responsive to aging and that expression of the neuron specific PGC-1a isoform allows for specialization in metabolic adaptation. Transcriptional profiles of the cortex from male mice show an impact of age on immune, inflammatory, and neuronal functional pathways and a highly integrated metabolic response that is associated with decreased expression of PGC-1a. Proteomic analysis confirms age-related changes in metabolism and further shows changes in ribosomal and RNA splicing pathways. We show that neurons express a specialized PGC-1a isoform that becomes active during differentiation from stem cells and is further induced during the maturation of isolated neurons. Neuronal but not astrocyte PGC-1a responds robustly to inhibition of the growth sensitive kinase GSK3b, where the brain specific promoter driven dominant isoform is repressed. The GSK3b inhibitor lithium broadly reprograms metabolism and growth signaling, including significantly lower expression of mitochondrial and ribosomal pathway genes and suppression of growth signaling, which are linked to changes in mitochondrial function and neuronal outgrowth. In vivo, lithium treatment significantly changes the expression of genes involved in cortical growth, endocrine, and circadian pathways. These data place the GSK3b/PGC-1a axis centrally in a growth and metabolism network that is directly relevant to brain aging.

Fluorescent tagging of endogenous proteins with CRISPR/Cas9 in primary mouse neural stem cells.

Although exogenous overexpression of a protein fused to a fluorescent tag can provide insight for the protein's function, it also can produce artifacts attributed to its upregulation and may not fully report the endogenous regulation of the protein of interest. To circumvent these issues, we adapted a protocol to label endogenous proteins with fluorescent tags in primary adult mouse neural stem cells in vitro. Here, we describe reagent construction, reagent delivery, and a screening strategy to isolate edited cells. For complete details on the use and execution of this protocol, please refer to Morrow et al. (2020).

Vimentin Coordinates Protein Turnover at the Aggresome during Neural Stem Cell Quiescence Exit.

Maintaining a healthy proteome throughout life is critical for proper somatic stem cell function, but the complexities of the stem cell response to increases in damaged or aggregated proteins remain unclear. Here we demonstrate that adult neural stem cells (NSCs) utilize aggresomes to recover from disrupted proteostasis and describe a novel function for the intermediate filament vimentin in proteostasis as a spatial coordinator of proteasomes to the aggresome. In the absence of vimentin, NSCs have a reduced capacity to exit quiescence, a time when NSCs are required to clear a wave of aggregated proteins, and demonstrate an early age-dependent decline in proliferation and neurogenesis. Taken together, these data reveal a significant role of vimentin and aggresomes in the regulation of proteostasis during quiescent NSC activation.