Autofluorescence lifetime flow cytometry with time-correlated single photon counting.

Autofluorescence lifetime imaging microscopy (FLIM) is sensitive to metabolic changes in single cells based on changes in the protein-binding activities of the metabolic co-enzymes NAD(P)H. However, FLIM typically relies on time-correlated single-photon counting (TCSPC) detection electronics on laser-scanning microscopes, which are expensive, low-throughput, and require substantial post-processing time for cell segmentation and analysis. Here, we present a fluorescence lifetime-sensitive flow cytometer that offers the same TCSPC temporal resolution in a flow geometry, with low-cost single-photon excitation sources, a throughput of tens of cells per second, and real-time single-cell analysis. The system uses a 375nm picosecond-pulsed diode laser operating at 50MHz, alkali photomultiplier tubes, an FPGA-based time tagger, and can provide real-time phasor-based classification ( i.e ., gating) of flowing cells. A CMOS camera produces simultaneous brightfield images using far-red illumination. A second PMT provides two-color analysis. Cells are injected into the microfluidic channel using a syringe pump at 2-5 mm/s with nearly 5ms integration time per cell, resulting in a light dose of 2.65 J/cm 2 that is well below damage thresholds (25 J/cm 2 at 375 nm). Our results show that cells remain viable after measurement, and the system is sensitive to autofluorescence lifetime changes in Jurkat T cells with metabolic perturbation (sodium cyanide), quiescent vs. activated (CD3/CD28/CD2) primary human T cells, and quiescent vs. activated primary adult mouse neural stem cells, consistent with prior studies using multiphoton FLIM. This TCSPC-based autofluorescence lifetime flow cytometer provides a valuable label-free method for real-time analysis of single-cell function and metabolism with higher throughput than laser-scanning microscopy systems.

Classification of Neural Stem Cell Activation State In Vitro using Autofluorescence.

Neural stem cells (NSCs) divide and produce newborn neurons in the adult brain through a process called adult neurogenesis. Adult NSCs are primarily quiescent, a reversible cell state where they have exited the cell cycle (G0) yet remain responsive to the environment. In the first step of adult neurogenesis, quiescent NSCs (qNSCs) receive a signal and activate, exiting quiescence and re-entering the cell cycle. Thus, understanding the regulators of NSC quiescence and quiescence exit is critical for future strategies targeting adult neurogenesis. However, our understanding of NSC quiescence is limited by technical constraints in identifying quiescent NSCs (qNSCs) and activated NSCs (aNSCs). This protocol describes a new approach to identify and enrich qNSCs and aNSCs generated in in vitro cultures by imaging NSC autofluorescence. First, this protocol describes how to use a confocal microscope to identify autofluorescent markers of qNSCs and aNSCs to classify NSC activation state using autofluorescence intensity. Second, this protocol describes how to use a fluorescent activated cell sorter (FACS) to classify NSC activation state and enrich samples for qNSCs or aNSCs using autofluorescence intensity. Third, this protocol describes how to use a multiphoton microscope to perform fluorescence lifetime imaging (FLIM) at single-cell resolution, classify NSC activation state, and track the dynamics of quiescent exit using both autofluorescence intensities and fluorescence lifetimes. Thus, this protocol provides a live-cell, label-free, single-cell resolution toolkit for studying NSC quiescence and quiescence exit.

Autofluorescence is a biomarker of neural stem cell activation state.

Neural stem cells (NSCs) must exit quiescence to produce neurons; however, our understanding of this process remains constrained by the technical limitations of current technologies. Fluorescence lifetime imaging (FLIM) of autofluorescent metabolic cofactors has been used in other cell types to study shifts in cell states driven by metabolic remodeling that change the optical properties of these endogenous fluorophores. Using this non-destructive, live-cell, and label-free strategy, we found that quiescent NSCs (qNSCs) and activated NSCs (aNSCs) have unique autofluorescence profiles. Specifically, qNSCs display an enrichment of autofluorescence localizing to a subset of lysosomes, which can be used as a graded marker of NSC quiescence to predict cell behavior at single-cell resolution. Coupling autofluorescence imaging with single-cell RNA sequencing, we provide resources revealing transcriptional features linked to deep quiescence and rapid NSC activation. Together, we describe an approach for tracking mouse NSC activation state and expand our understanding of adult neurogenesis.

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.

Moving CNS axon growth and regeneration research into human model systems.

Axon regeneration is limited in the adult mammalian central nervous system (CNS) due to both intrinsic and extrinsic factors. Rodent studies have shown that developmental age can drive differences in intrinsic axon growth ability, such that embryonic rodent CNS neurons extend long axons while postnatal and adult CNS neurons do not. In recent decades, scientists have identified several intrinsic developmental regulators in rodents that modulate growth. However, whether this developmentally programmed decline in CNS axon growth is conserved in humans is not yet known. Until recently, there have been limited human neuronal model systems, and even fewer age-specific human models. Human in vitro models range from pluripotent stem cell-derived neurons to directly reprogrammed (transdifferentiated) neurons derived from human somatic cells. In this review, we discuss the advantages and disadvantages of each system, and how studying axon growth in human neurons can provide species-specific knowledge in the field of CNS axon regeneration with the goal of bridging basic science studies to clinical trials. Additionally, with the increased availability and quality of 'omics datasets of human cortical tissue across development and lifespan, scientists can mine these datasets for developmentally regulated pathways and genes. As there has been little research performed in human neurons to study modulators of axon growth, here we provide a summary of approaches to begin to shift the field of CNS axon growth and regeneration into human model systems to uncover novel drivers of axon growth.

Age-maintained human neurons demonstrate a developmental loss of intrinsic neurite growth ability.

Injury to adult mammalian central nervous system (CNS) axons results in limited regeneration. Rodent studies have revealed a developmental switch in CNS axon regenerative ability, yet whether this is conserved in humans is unknown. Using human fibroblasts from 8 gestational-weeks to 72 years-old, we performed direct reprogramming to transdifferentiate fibroblasts into induced neurons (Fib-iNs), avoiding pluripotency which restores cells to an embryonic state. We found that early gestational Fib-iNs grew longer neurites than all other ages, mirroring the developmental switch in regenerative ability in rodents. RNA-sequencing and screening revealed ARID1A as a developmentally-regulated modifier of neurite growth in human neurons. These data suggest that age-specific epigenetic changes may drive the intrinsic loss of neurite growth ability in human CNS neurons during development. One-Sentence Summary: Directly-reprogrammed human neurons demonstrate a developmental decrease in neurite growth ability.

Neural stem cell metabolism revisited: a critical role for mitochondria.

Metabolism has emerged as a key regulator of stem cell behavior. Mitochondria are crucial metabolic organelles that are important for differentiated cells, yet considered less so for stem cells. However, recent studies have shown that mitochondria influence stem cell maintenance and fate decisions, inviting a revised look at this topic. In this review, we cover the current literature addressing the role of mitochondrial metabolism in mouse and human neural stem cells (NSCs) in the embryonic and adult brain. We summarize how mitochondria are implicated in fate regulation and how substrate oxidation affects NSC quiescence. We further explore single-cell RNA sequencing (scRNA-seq) data for metabolic signatures of adult NSCs, highlight emerging technologies reporting on metabolic signatures, and discuss mitochondrial metabolism in other stem cells.

Adult fibroblasts use aggresomes only in distinct cell-states.

The aggresome is a protein turnover system in which proteins are trafficked along microtubules to the centrosome for degradation. Despite extensive focus on aggresomes in immortalized cell lines, it remains unclear if the aggresome is conserved in all primary cells and all cell-states. Here we examined the aggresome in primary adult mouse dermal fibroblasts shifted into four distinct cell-states. We found that in response to proteasome inhibition, quiescent and immortalized fibroblasts formed aggresomes, whereas proliferating and senescent fibroblasts did not. Using this model, we generated a resource to provide a characterization of the proteostasis networks in which the aggresome is used and transcriptomic features associated with the presence or absence of aggresome formation. Using this resource, we validate a previously reported role for p38 MAPK signaling in aggresome formation and identify TAK1 as a novel driver of aggresome formation upstream of p38 MAPKs. Together, our data demonstrate that the aggresome is a non-universal protein degradation system which can be used cell-state specifically and provide a resource for studying aggresome formation and function.

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).

Declining lamin B1 expression mediates age-dependent decreases of hippocampal stem cell activity.

Neural stem cells (NSCs) generate neurons throughout life in the hippocampal dentate gyrus. With advancing age, levels of neurogenesis sharply drop, which has been associated with a decline in hippocampal memory function. However, cell-intrinsic mechanisms mediating age-related changes in NSC activity remain largely unknown. Here, we show that the nuclear lamina protein lamin B1 (LB1) is downregulated with age in mouse hippocampal NSCs, whereas protein levels of SUN-domain containing protein 1 (SUN1), previously implicated in Hutchinson-Gilford progeria syndrome (HGPS), increase. Balancing the levels of LB1 and SUN1 in aged NSCs restores the strength of the endoplasmic reticulum diffusion barrier that is associated with segregation of aging factors in proliferating NSCs. Virus-based restoration of LB1 expression in aged NSCs enhances stem cell activity in vitro and increases progenitor cell proliferation and neurogenesis in vivo. Thus, we here identify a mechanism that mediates age-related decline of neurogenesis in the mammalian hippocampus.

Vimentin's side gig: Regulating cellular proteostasis in mammalian systems.

Intermediate filaments (IFs) perform a diverse set of well-known functions including providing structural support for the cell and resistance to mechanical stress, yet recent evidence has revealed unexpected roles for IFs as stress response proteins. Previously, it was shown that the type III IF protein vimentin forms cage-like structures around centrosome-associated proteins destined for degradation, structures referred to as aggresomes, suggesting a role for vimentin in protein turnover. However, vimentin's function at the aggresome has remained largely understudied. In a recent report, vimentin was shown to be dispensable for aggresome formation, but played a critical role in protein turnover at the aggresome through localizing proteostasis-related machineries, such as proteasomes, to the aggresome. Here, we review evidence for vimentin's function in proteostasis and highlight the organismal implications of these findings.

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.

Stem Cell Aging? Blame It on the Niche.

The molecular basis for the neural stem cell quiescence-to-activation transition has become an important focus in the study of adult neurogenesis. Recently in Cell, Kalamakis et al. (2019) show that aged neural stem cells face greater barriers to exiting quiescence, imposed by the niche through inflammation and altered Wnt signaling.

The Krüppel-Like Factor Gene Target Dusp14 Regulates Axon Growth and Regeneration.

Purpose: Adult central nervous system (CNS) neurons are unable to regenerate their axons after injury. Krüppel-like transcription factor (KLF) family members regulate intrinsic axon growth ability in vitro and in vivo, but mechanisms downstream of these transcription factors are not known. Methods: Purified retinal ganglion cells (RGCs) were transduced to express exogenous KLF9, KLF16, KLF7, or KLF11; microarray analysis was used to identify downstream genes, which were screened for effects on axon growth. Dual-specificity phosphatase 14 (Dusp14) was further studied using genetic (siRNA, shRNA) and pharmacologic (PTP inhibitor IV) manipulation to assess effects on neurite length in vitro and survival and regeneration in vivo after optic nerve crush in rats and mice. Results: By screening genes regulated by KLFs in RGCs, we identified Dusp14 as a critical gene target limiting axon growth and regeneration downstream of KLF9's ability to suppress axon growth in RGCs. The KLF9-Dusp14 pathway inhibited activation of mitogen-activated protein kinases normally critical to neurotrophic signaling of RGC survival and axon elongation. Decreasing Dusp14 expression or disrupting its function in RGCs increased axon growth in vitro and promoted survival and optic nerve regeneration after optic nerve injury in vivo. Conclusions: These results link intrinsic and extrinsic regulators of axon growth and suggest modulation of the KLF9-Dusp14 pathway as a potential approach to improve regeneration in the adult CNS after injury.

KLF9 and JNK3 Interact to Suppress Axon Regeneration in the Adult CNS.

Neurons in the adult mammalian CNS decrease in intrinsic axon growth capacity during development in concert with changes in Krüppel-like transcription factors (KLFs). KLFs regulate axon growth in CNS neurons including retinal ganglion cells (RGCs). Here, we found that knock-down of KLF9, an axon growth suppressor that is normally upregulated 250-fold in RGC development, promotes long-distance optic nerve regeneration in adult rats of both sexes. We identified a novel binding partner, MAPK10/JNK3 kinase, and found that JNK3 (c-Jun N-terminal kinase 3) is critical for KLF9's axon-growth-suppressive activity. Interfering with a JNK3-binding domain or mutating two newly discovered serine phosphorylation acceptor sites, Ser106 and Ser110, effectively abolished KLF9's neurite growth suppression in vitro and promoted axon regeneration in vivo These findings demonstrate a novel, physiologic role for the interaction of KLF9 and JNK3 in regenerative failure in the optic nerve and suggest new therapeutic strategies to promote axon regeneration in the adult CNS.SIGNIFICANCE STATEMENT Injured CNS nerves fail to regenerate spontaneously. Promoting intrinsic axon growth capacity has been a major challenge in the field. Here, we demonstrate that knocking down Krüppel-like transcription factor 9 (KLF9) via shRNA promotes long-distance axon regeneration after optic nerve injury and uncover a novel and important KLF9-JNK3 interaction that contributes to axon growth suppression in vitro and regenerative failure in vivo These studies suggest potential therapeutic approaches to promote axon regeneration in injury and other degenerative diseases in the adult CNS.

A Fatty Acid Oxidation-Dependent Metabolic Shift Regulates Adult Neural Stem Cell Activity.

Hippocampal neurogenesis is important for certain forms of cognition, and failing neurogenesis has been implicated in neuropsychiatric diseases. The neurogenic capacity of hippocampal neural stem/progenitor cells (NSPCs) depends on a balance between quiescent and proliferative states. Here, we show that the rate of fatty acid oxidation (FAO) regulates the activity of NSPCs. Quiescent NSPCs show high levels of carnitine palmitoyltransferase 1a (Cpt1a)-dependent FAO, which is downregulated in proliferating NSPCs. Pharmacological inhibition and conditional deletion of Cpt1a in vitro and in vivo leads to altered NSPC behavior, showing that Cpt1a-dependent FAO is required for stem cell maintenance and proper neurogenesis. Strikingly, manipulation of malonyl-CoA, the metabolite that regulates levels of FAO, is sufficient to induce exit from quiescence and to enhance NSPC proliferation. Thus, the data presented here identify a shift in FAO metabolism that governs NSPC behavior and suggest an instructive role for fatty acid metabolism in regulating NSPC activity.

Role of Mitochondrial Metabolism in the Control of Early Lineage Progression and Aging Phenotypes in Adult Hippocampal Neurogenesis.

Precise regulation of cellular metabolism is hypothesized to constitute a vital component of the developmental sequence underlying the life-long generation of hippocampal neurons from quiescent neural stem cells (NSCs). The identity of stage-specific metabolic programs and their impact on adult neurogenesis are largely unknown. We show that the adult hippocampal neurogenic lineage is critically dependent on the mitochondrial electron transport chain and oxidative phosphorylation machinery at the stage of the fast proliferating intermediate progenitor cell. Perturbation of mitochondrial complex function by ablation of the mitochondrial transcription factor A (Tfam) reproduces multiple hallmarks of aging in hippocampal neurogenesis, whereas pharmacological enhancement of mitochondrial function ameliorates age-associated neurogenesis defects. Together with the finding of age-associated alterations in mitochondrial function and morphology in NSCs, these data link mitochondrial complex function to efficient lineage progression of adult NSCs and identify mitochondrial function as a potential target to ameliorate neurogenesis-defects in the aging hippocampus.