Partial reprogramming of the mammalian brain

Abstract

Xu and colleagues used partial OSKM reprogramming in aged mice to drive cell-type proportions of the subventricular zone to more youthful levels, which equates to qualified rejuvenation of a neurogenic niche that is defined, in part, by restoration of neuroblast levels.

For generations, humans have sought to understand why we age and how to manipulate aging processes in pursuit of longer, healthier lives. Our advancing ability to measure these processes is partially reflected in emerging biological clocks. These clocks function under the premise that aging is not simply a chronological process; the variable manifestations of aging can be quantified to determine relative biological age^1,2. Having detached the age of an organism from the basic passing of time, we are faced with the challenge of beginning to unwind and reset biological clocks. One promising approach to this challenge is using reprogramming to rejuvenate cells and tissues.

In this issue of Nature Aging, Xu and colleagues explored the effects of partial reprogramming in the aged-brain subventricular zone (SVZ)³. They convincingly demonstrate the ability to induce a more youthful SVZ cellular composition following partial reprogramming throughout the entire mouse or specifically in the SVZ. Through in-depth analysis of single-cell sequencing data and immunofluorescent imaging, they mapped the cell types and affected pathways that are responsible for turning back the aging clock in the SVZ.

Xu and colleagues’ discoveries follow a groundbreaking study by Takahashi and Yamanaka in 2006 that demonstrated that the collective expression of four factors, encoded by Oct4, Sox2, Klf4 and Myc (the OSKM genes), can reprogram cells to become induced pluripotent stem cells, effectively resetting the biological clock on a cellular level⁴. Expressing OSKM genes was quickly investigated in vivo, and it was found that although resetting the clock was possible, the loss of cellular somatic identity was problematic and resulted in increased apoptosis and tumorigenesis^5,6. Mice that undergo whole-body expression of OSKM genes typically die within weeks of constitutive gene expression⁷.

Several strategies are under development for overcoming these challenges. Direct reprogramming exploits unique sets of factors or small molecules to induce desired transdifferentiation, thereby bypassing the stem cell stage involved in induced pluripotent stem cell reprogramming⁸. Partial reprogramming, as used by Xu and colleagues, involves repeated transient expression of the OSKM genes to generate intermediates that retain cellular identity, thereby avoiding tumorigenesis, while mitigating age-associated changes (such as DNA damage and senescence)^9,10.

Although rejuvenation of any aged tissue could offer tremendous therapeutic value, winding back the clock on the aging brain is a particularly important experimental goal. At least in concept, reprogramming the aged mammalian brain to a more youthful state could preserve late-life cognitive function and potentially stave off neurodegenerative conditions that affect millions of people globally. Cellular niches that possess intrinsic regenerative capacity lost in aging are intuitive foci for cellular reprogramming. Neurogenesis (the ability to generate new neurons) occurs in select brain regions, including the SVZ, and declines sharply in aging. Reductions in proliferating neural stem cells (NSCs) and the neuroblasts they generate, as well as an increased propensity for NSCs to generate glia, underlie the loss of neurogenic potential in aging^11,12.

The authors first implemented whole-body reprogramming using pulsed expression of OSKM genes in old mice (18–20 or 24–26 months old). This resulted in a partial reversal of the age-associated decline in SVZ neuroblast abundance without significantly affecting the proportion of neuroblast precursor cells, which suggests a shift in NSC differentiation or neuroblast survival and not simply an increase in proliferation (Fig. 1). Notably, the authors did not observe production of new or undifferentiated cell types in the SVZ. Through transcriptomic analysis of individual cell types, they observed a reversal of aging signatures (including cell adhesion molecule binding in neuronal progenitors), but they also observed exacerbation of aging signatures (including an inflammatory response in neuroblasts).

Fig. 1 |. Partial OSKM reprogramming reverts aged SVZ cell composition toward a more youthful state.

Xu et al. demonstrate that whole-body and SVZ-localized partial reprogramming partially restored the abundance of SVZ neuroblasts toward youthful levels. SVZ-localized partial reprogramming reverted age-dependent declines in NPCs and exacerbated age-related decline in astrocytes. Population shifts were observed in other cell types within the SVZ, including endothelial and immune cells. Partial rejuvenation of neuroblasts and NPC proportions indicate potential rejuvenation of the neurogenic niche. Cell proportion data displayed in the stacked bar chart reflect average cell type proportions for each condition from extended data figures 2h and 6a of Xu et al. Figure created with BioRender.com.

To delineate region-specific effects, they implemented a mouse model in which stereotaxic viral injection of Cre recombinase into the lateral ventricle mediates expression of the reverse tetracycline transactivator and targeted partial reprogramming of cells in the SVZ. SVZ-localized reprogramming enabled longer pulse activations of OSKM genes without observable consequences to the general health of the mice. Upon examining the cell-type proportions following localized reprogramming, they observed significant increases in neuroblasts and neuronal precursor cells in old mice (26–28 months old) to levels that approached those of young mice. Cell–cell adhesion in neuronal precursors and neuroblasts, and neuronal migration in neuroblasts, were among the age-altered transcriptional signatures that were reversed by partial reprogramming. The authors also observed elevated infiltrating immune cells in the SVZ, which was pronounced in young mice and coincided with increased inflammatory signatures in several cell types. Increased infiltrating immune cells is a likely consequence, at least in part, of the surgery required in this model. However, inflammatory activation in both models warrants further examination.

The authors implemented two algorithms, a transcriptomic-based aging clock¹ and a linear regression model based on cell-type proportions, to estimate the relative age of the SVZ using single-cell sequencing data. Transcriptomic clocks that assessed the age of individual cell types tended to find no change or an apparent increase in age following partial reprogramming. Using the linear regression model to determine the relative age of the SVZ, the authors observed a noteworthy median rejuvenation of 10.9 months following SVZ-targeted reprogramming. Discrepancy between the two prediction models may reflect higher heterogeneity in the transcriptomic signatures compared to the cell proportion data.

Across the two reprogramming models, youthful characteristics of the SVZ tissue were most consistently attributed to increased neuroblast proportions. Thus, Xu et al. turned to in vitro reprogramming to better understand the effects of OSKM expression on NSC differentiation, which gives rise to neuroblasts. Reprogramming of NSCs in culture under differentiating conditions recapitulated the increase in neuroblast cells observed in vivo without increasing NSC proliferation. This reinforced the notion that increased neuroblast abundance probably results from reversal of glial skew in aging, or perhaps from improved neuroblast survival following partial reprogramming. The authors identified Lrfn4 and Brd2 among age-altered reprogrammed genes that were restored to more youthful levels. Lrfn4 is involved in the regulation of neurite outgrowth¹³, and Brd2 is expressed in proliferating NSCs and can block differentiation when overexpressed¹⁴. Both genes were expressed at higher levels in old NSC cultures and reduced following reprogramming. Critically, the authors demonstrated that reprogramming and differentiating old NSCs in vitro increased the production of neurons and, similarly, in vivo reprogramming boosted the number of new neurons in the old olfactory bulb, the region to which SVZ-born neuroblasts migrate.

Single-cell sequencing of the SVZ following whole-body and region-specific partial reprogramming enabled in-depth analysis and comparison of SVZ cellular composition changes. Aged cell proportion and transcriptional signatures, as well as overall SVZ age (via the linear model), tended to be more robustly reverted under the localized protocol, which suggests that targeted approaches may offer better reprogramming control than whole-body approaches. However, it remains to be seen how tissue-specific and interorgan systems adapt to localized versus systemic reprogramming. Both the reversed and exacerbated aging profiles discovered in response to partial reprogramming are valuable; these positive and negative consequences are vital comparators in forthcoming iterative protocols aimed at optimizing partial reprogramming.

Xu and colleagues’ findings build on previous investigations of brain reprogramming, including a study by Rodríguez-Matellán and colleagues that demonstrated that cycled, whole-body expression of OSKM genes in ten-month-old mice led to a greater migration of doublecortin-positive cells in the hippocampal dentate gyrus (another site of adult neurogenesis), as well as improvements in an object recognition memory task¹⁵. Collectively, emerging results demonstrate promise for partial reprogramming as a strategy to revert properties of aging-brain neurogenic zones to more youthful states.

The long-term consequences of partial reprogramming in the SVZ and other brain regions, including reprogramming stability and effects on individual circuits and cognitive domains, are important areas of future discovery that are well-recognized by Xu and colleagues. Lifespan studies following partial reprogramming will be critical for comprehensively characterizing systemic benefits and adverse effects. Whether and how small-molecule approaches can be integrated with genetic reprogramming to maximize rejuvenation and mitigate detrimental outcomes is an important future direction.

The seminal discoveries by Xu and colleagues emphasize the potential for applying partial reprogramming to revert age-vulnerable brain microenvironments to more youthful states and also raise important questions that will need to be answered regarding the safety and stability of OSKM manipulation under distinct conditions. Only time will tell whether this research trajectory someday leads to translational interventions that preserve long-term health and function of the human brain.