Enhanced NRF2 expression mitigates the decline in neural stem cell function during aging

Abstract Although it is known that aging affects neural stem progenitor cell (NSPC) biology in fundamental ways, the underlying dynamics of this process are not fully understood. Our previous work identified a specific critical period (CP) of decline in NSPC activity and function during middle age (13–15 months), and revealed the reduced expression of the redox‐sensitive transcription factor, NRF2, as a key mediator of this process. Here, we investigated whether augmenting NRF2 expression could potentially mitigate the NSPC decline across the identified CP. NRF2 expression in subventricular zone (SVZ) NSPCs was upregulated via GFP tagged recombinant adeno‐associated viral vectors (AAV‐NRF2‐eGFP), and its cellular and behavioral effects compared to animals that received control vectors (AAV‐eGFP). The vectors were administered into the SVZs of aging rats, at time points either before or after the CP. Results indicate that animals that had received AAV‐NRF2‐eGFP, prior to the CP (11 months of age), exhibited substantially improved behavioral function (fine olfactory discrimination and motor tasks) in comparison to those receiving control viruses. Further analysis revealed that NSPC proliferation, self‐renewal, neurogenesis, and migration to the olfactory bulb had significantly increased upon NRF2 upregulation. On the other hand, increasing NRF2 after the CP (at 20 months of age) produced no notable changes in NSPC activity at either cellular or behavioral levels. These results, for the first time, indicate NRF2 pathway modulation as a means to support NSPC function with age and highlight a critical time‐dependency for activating NRF2 to enhance NSPC function.

. Given the pivotal role of stem cells in tissues with lifelong regenerative capacity such as the brain, understanding stem cell aging will be important if we are to understand aging at the organ level. More broadly, comprehending stem cell aging will also support the development of interventions that could improve both health and lifespan.
In this context, our previous studies, conducted in naturally aging rodents, identified a specific temporal pattern of change in NSPC dynamics during aging. In particular, the studies highlighted a critical time during middle age (13-15 months), when the regenerative function of NSPCs showed a striking decline (Corenblum et al., 2016;Ray et al., 2018;Schmidlin et al., 2019). The studies also determined the reduced expression of nuclear factor (erythroid-derived 2) like 2 (or NRF2), as a key mechanism mediating this phenomenon. As such, this work provided first evidence of an important regulatory role for NRF2 in NSPC aging.
NRF2 is a redox-sensitive transcription factor known to be essential to the cell's homeostatic mechanism (Bryan et al., 2013;Itoh et al., 2010;Suzuki & Yamamoto, 2017). NRF2 is ubiquitously expressed in most eukaryotic cells and functions to induce a broad range of cellular defenses against exogenous and endogenous stresses, including oxidants, xenobiotics, inflammatory agents, and excessive nutrient/metabolite supply. In particular, NRF2 can up-regulate a range of classical ARE (antioxidant response element)-driven genes, encoding major antioxidants and other detoxification enzymes. In addition to its classical function in regulating the stress response, NRF2 has been linked to cell growth, proliferation, mitochondrial and trophic functions, protein quality control, and increased lifespan (Holmstrom et al., 2013;Malhotra et al., 2010;Sykiotis & Bohmann, 2008;Tullet et al., 2008;Wakabayashi et al., 2010;Wiesner et al., 2013;Zhu et al., 2013). Our recent work adds a unique and important new face to NRF2 actions in the cell-namely the age-relevant regulation of NSPCs (Corenblum et al., 2016;Madhavan, 2015;Ray et al., 2018;Schmidlin et al., 2019).
Given that NRF2 loss accentuates NSPC aging, in this study, we investigated whether increasing NRF2 levels could boost NSPC function with age. In particular, we studied whether inducing high intrinsic NRF2 expression can potentially mitigate the decline in NSPC regeneration during the critical middle-age period between 13 and 15 months (mos), identified in our previous work. NRF2 was delivered to rat subventricular zone (SVZ) NSPCs through recombinant adeno-associated viral (AAV) vectors injected either before (at 11 mos of age) or well after the critical aging period (at 20 mos of age). We find that the administration of AAV-NRF2-eGFP vectors before the initiation of the critical period (CP), substantially improved SVZ NSPC regeneration and associated behavioral function, as compared to controls (AAV-eGFP delivery). On the other hand, application of AAV-NRF2-eGFP after the conclusion of the CP failed to significantly promote NSPC activity and function.
These data establish a major governing role for NRF2 in NSPCs and support targeting the NRF2 pathway as a potential approach to advantageously modulate NSPC function with age.

| Viral expression of NRF2 in aging SVZ NSPCs improves behavioral function during the critical period
In order to address whether augmenting NRF2 expression can promote NSPC function during aging, recombinant adeno-associated viral vectors tagged with a GFP reporter carrying either NRF2 (AAV-NRF2-eGFP), or eGFP alone (AAV-eGFP) as a mock control, were stereotactically delivered into the SVZs of aging rats. To specifically determine the effects of rescuing NRF2 expression in the context of the critical middle-age period (13-15 mos) identified in our previous studies, the vectors were injected into the SVZ either before (11 mos of age) or well after (20 mos of age) the CP. Subsequently, behavioral function (at 2 and 4 mos post-viral injection) and cellular changes (4 mos post-injection) were assessed ( Figure 1a).
First, we confirmed the efficiency of viral transduction. It was found that AAV2/1 administration into two sites along the rostrocaudal extent of the lateral SVZ robustly and specifically transduces NSPCs (stereotaxic locations shown in Figure S1A,C and described in the Methods section). Strong GFP expression was noted in the rat SVZ by immunofluorescence microscopy (Figure 1b, broader views of the transduced areas are in Figure S1B,D). This high GFP expression was seen as early as 2 weeks post-injection, with peak viral transduction reached at 1.5 mos. As shown, co-labeling with antibodies targeting the NSPC specific antigen Musashi1 (expressed by a large population of SVZ stem and progenitor cells) indicated that AAV2/1 proficiently infected SVZ NSPCs (confocal micrographs in Figure 1A-C). Moreover, significantly increased NRF2 expression was seen in SVZ cells of animals that received AAV-NRF2-eGFP, as compared to GFP controls ( Figure 1D-I). To ensure that NRF2 overexpression further activates downstream target genes, levels of the well-established NRF2 target gene, glutamate-cysteine ligase modifier subunit (GCLM), in the SVZ were also assessed. As shown, GCLM expression was increased in the same SVZ cells that showed high NRF2 expression thus confirming NRF2 pathway activation ( Figure 1J-Q). This level of NRF2 expression and activation appeared comparable to what was observed in 9-to 11-month-old animals as characterized previously (Corenblum et al., 2016).
Next, the behavioral consequences of increased NRF2 expression were analyzed. The fine olfactory discrimination task is a known measure of SVZ NSPC function that tests the animal's ability to discriminate between different ratios of [+]/good tasting coconut (COC) and [−]/bad tasting mixture of almond and denatonium benzoate (ALM) (Corenblum et al., 2016;Enwere et al., 2004;Schmidlin et al., 2019). As expected, the baseline olfactory function (i.e., prior to AAV injection) of the older 20 mos rats was significantly worse (reflected by lower scores on the Y-axis) than the 11-month-old animals (Figure 2A,D). Intriguingly, as compared to the AAV-eGFP control-injected rats, the 11-month-old animals that received AAV-NRF2-GFP exhibited an increased capacity to discriminate between very similar ratios of COC and ALM (56:44) starting at 2 mos after injection [ Figure 2B; p = 0.011, F 3,33 = 217.124 (concentration), two-way RM-ANOVA], which became even more significant ( We also assessed motor function via a challenging beam task to investigate potential striatal effects of increased SVZ NRF2 expression. We generated a composite score that represents the ability of an animal to cross an increasingly narrow in size set of beams without foot slip errors, scooting across, or failing to cross the beam ( Figure 2G-R). While there was no significant difference in the composite scores between animals at 2 mos post-AAV injection, the 11-month-old animals injected with AAV-NRF2-eGFP were able to successfully traverse both the 20 mm and 15 mm beams more often than their AAV-eGFP-injected counterparts at the 4 mos postinjection time point ( Figure 2I; 20 mm beam -p = 0.0395, unpaired t test, t = 2.20, df = 20; 15 mm beam -p = 0.028, unpaired t test, t = 2.29, df = 31). Interestingly, AAV-NRF2-eGFP-injected rats also F I G U R E 1 NRF2 expression and activation in the SVZ NSPCs. (a) Depicts the experimental design and timeline. (b) Shows GFP expression in SVZ cells 1 mos after AAV-eGFP injection (white arrows). A-C Depicts confocal images of Musashi + NSPCs showing high GFP expression after AAV-eGFP transduction (arrows indicate example positive cells). GFP expressing NSPCs in the dorsolateral SVZ showed increased NRF2 expression in AAV-Nr2-eGFP-injected rats (G-I, arrows) compared to rats that had received AAV-eGFP (D-F). The NRF2 target gene, GCLM, was highly expressed in the same NRF2 overexpressing cells of AAV-NRF2-eGFP rats (N-Q) compared to control rats (J-M). Inset in O shows higher magnification view of NRF2/GCLM co-labeling. Scale bar of 20 μM, applicable to images in A-Q, is drawn in Q activation is delayed to an older age after the completion of the critical period.

| SVZ NSPC proliferation and neurogenesis are enhanced following NRF2 overexpression during the critical period
Based on our findings that increased NRF2 expression can promote SVZ-associated behavioral function, we next investi-

| Increased NRF2 supports SVZ NSPC regeneration during the critical period
Given that viral NRF2 expression during the critical period increased SVZ NSPC proliferation and neurogenesis, we interrogated how NRF2 affects various NPSC subtypes in the SVZ by examining the expression of markers that delineate different SVZ stem and progenitor cells. SVZ NSPCs show a hierarchy of division: glial-like type B cells divide relatively infrequently to give rise to rapidly dividing type C transit-amplifying cells (also referred to as intermediate progenitor cells), which expand the progenitor pool. These type C transit-amplifying cells then generate immature type A neuroblasts that mature into fully differentiated neurons. To assess these different NSPC subtypes, we first stained for Musashi1 (Mus) that is highly expressed in type B and type C NSPCs. It was observed that Mus immunolabeling (red) in 11-month-old AAV-NRF2-eGFP-injected rats was notably greater than in AAV-eGFP controls ( Figure 4A-F).
Confocal quantification confirmed higher numbers of Mus + cells in the dorsolateral SVZ of AAV-NRF2-eGFP-injected rats compared to F I G U R E 2 Heightened olfactory discrimination and motor abilities in NRF2 overexpressing rats. Results from baseline testing of fine olfactory discrimination (upper schematic on the left) on naïve 11-month and 20-month rats (aging stages before and after the CP are in (A) and (D). 11-month-old rats showed significantly improved abilities to discriminate between similar concentrations of odorants, 2 mos (B) and 4 mos (C) after AAV-NRF2-eGFP administration, compared to controls. Analysis of rats which received AAV-NRF2-eGFP at 20 mos of age (after the CP) showed no positive effect on fine olfactory discrimination capacities (E,F). "Ratio of odor components" label below (D-F) graphs applies to all six olfactory graphs above. [*p < 0.05, **p < 0.01, Two-way repeated measures ANOVA with Tukey's post hoc test].
Lower schematic on the left shows the challenging beam apparatus. Younger 11-month-old rats showed similar composite motor scores at baseline (G) and 2 mos (H) after AAV administration. However, rats receiving AAV-NRF2-eGFP displayed significantly higher composite scores at 4-month post-viral injection when traversing the 20 mm and 15 mm beams (I). (J-L) Shows beam traversal times for AAV-NRF2-eGFP rats and AAV-eGFP rats. AAV-NRF2-eGFP administration in older 20-month-old rats (after CP completion) had no effect on their composite motor scores when traversing the 30 mm beam at baseline (M), 2-month (N) or 4-month post-viral injection (O). Animals were unable to cross the 20 mm or 15 mm width beams (P,Q) at this age (indicated by "x"). Beam traversal time for the 20-month-old AAV-NRF2-eGFP-injected rats across the 30 mm beam is shown in (R). "Beam widths" label below (P-R) graphs applies to all nine beam graphs above.
[*p < 0.05, **p < 0.01, ***p < 0.001, unpaired t test with Welch's correction] controls ( Figure 4A; p = 0.003, unpaired t test, t = 4.72, df = 6). Next, we examined GFAP/Nestin (denotes type B NSPCs), Sox 2 (marker of C and some type B NSPCs), and Nestin (seen in type B and C NSPCs) expression ( Figure  These data, in concert with the data in Figure 3, suggest that NRF2 activation improves the regeneration of all major SVZ cell types, namely type B, type C and type A cells.

| Increased NRF2 promotes NSPC migration via the RMS during the critical period
Newly generated neuroblasts (type A cells) leave the SVZ from the anterior part (base of the anterior horn of the lateral ventricle) and  rRMS -p = 0.001, unpaired t test, t = 9.82, df = 6). These results suggested that newly generated NSPCs overexpressing NRF2 not only proliferate and regenerate at the level of the SVZ, but also migrate more effectively to the OB, than control cells. When NSPC migration was assessed in the older 20-month-old animals in the aSVZ and rRMS, it was found that the number of BrdU + /Dcx + expressing cells was higher on average in rats treated with AAV-NRF2-eGFP ( Figure   S2A-R). However, these increases were not statistically significant compared to control rats.

| Increased NRF2 expression supports NSPC differentiation and neuronal maturation
Having confirmed that an amplification of NRF2 expression improves NSPC proliferation, regeneration, and migration during the critical period, we studied whether these newly generated migratory suggesting that other cell-intrinsic and/or extrinsic factors maybe interfering with NRF2-mediated effects in the older animals.

| Increased NRF2 expression promotes striatal neurogenesis
Given the significant improvements in motor learning seen at 4 mos after viral NRF2 transduction in the 11-month-old rats, we also assessed striatal neurogenesis in the animals. Immunostaining with BrdU showed no apparent streams of potentially migrating cells from the subventricular zone in the AAV-NRF2-eGFP-injected animals, although occasional BrdU cells disjointed from the SVZ as well as pockets of BrdU cells in the striatum were noted (arrows in Figure 7A,B). The BrdU + cells were most often found as single isolated cells distributed predominantly in the dorsomedial, and some in the dorsolateral, striatum (schema in Figure 7C depicts this distribution of BrdU cells). NeuN immunostaining and highresolution confocal imaging showed that multiple BrdU cells were co-expressing the neuronal marker ( Figure 7D-K). Quantification determined that there were higher numbers of BrdU + /NeuN + double-stained cells in AAV-NRF2-eGFP-injected animals than in the animals that had received AAV-eGFP only ( Figure 7L). We also examined the differentiation of the newborn neurons into DARPP32 + cells, a marker of medium spiny neurons which constitute a large proportion of striatal neurons. However, we did not detect any BrdU labeled cells co-expressing DARPP32 ( Figure   S4A-H). These data indicated that increased NRF2 expression before the CP had induced striatal neurogenesis, but not their further subtype specification. before a certain critical period of vulnerability during aging, can enhance NSPC regeneration and function. These novel data are the F I G U R E 5 NSPCs travel more successfully through the rostral migratory stream to the olfactory bulb upon NRF2 upregulation. Immunohistochemical analysis of newborn BrdU + /Dcx + cells in the aSVZ, mRMS, and rRMS at 4 mos after viral injections in 15-month-old AAV-eGFP and AAV-Nr2-eGFP rats was conducted (see schematic at the top). AAV-NRF2-eGFP-injected animals showed higher BrdU/Dcx co-labeling in the aSVZ (A-H), mRMS (I-P), and rRMS (Q-X), compared to AAV-eGFP controls (Arrows point to example BrdU/Dcx doublepositive cells). Associated data from confocal quantification are shown in (a-c). (d) Conveys the average number of cells present across all three regions. [*p < 0.05, **p < 0.01, ***p < 0.001, unpaired t tests]. Scale bars: 10 μM. Scale bars for A-H is in H; for I-P is in P; for Q-X is in X first to reveal the ability of a cell-intrinsic factor, namely NRF2, to control NSPC aging and impact lifelong neural plasticity. Secondly, our data show that the observed activation of NSPC regeneration, upon NRF2 upregulation, correlated with a significantly better performance on fine olfactory discrimination and motor learning tasks-thus connecting molecular enhancements to a behaviorally relevant readout. It was noted that 11-month-old rats that had received control AAV viruses displayed an expected decline in olfactory discrimination function by 15 mos of age. However, rats that were administered AAVs encoding NRF2 exhibited superior olfactory discrimination abilities at 2 mos and 4 mos post-viral delivery (at 13 and 15 mos aging stages). This suggests that NRF2 upregulation can not only induce increased NSPC proliferation, self-renewal, differentiation, and migration to the OB, but also affect the olfactory circuitry leading to functional effects. In terms of motor function, AAV-NRF2-eGFP-injected rats showed higher composite motor scores and faster traverse times on a beam walking task. Specifically, NRF2 overexpressing animals were able to cross the narrower 20 and 15 mm beams without foot slips or falls, compared to control animals. The AAV-NRF2-eGFP animals also traveled across the 15 mm beam at a quicker pace than controls. These data suggest that NRF2based activation of NSPCs can also support striatum-based motor function. In this regard, it is known that a recruitment of newborn  (Benraiss et al., 2012;Kobayashi et al., 2006;Madhavan et al., 2012;Yamashita et al., 2006). Our data show that this was indeed the case in the NRF2 overexpressing rats, thus providing a basis for the improvement in motor learning in the 11-month-old animals.

| DISCUSS ION
A third important finding is that the supportive effects of NRF2 on NSPC activity and function were largely muted when viral NRF2 delivery was delayed until an older age of 20 mos (after the end of the CP). More specifically, although Musashi expressing cells were increased, and an almost significant increase in BrdU cell numbers (p = 0.052) was observed upon NRF2 upregulation in the older rats, other major NSPC populations (Nestin/GFAP, Nestin/Sox2, Dcx, and their behavior) were largely not affected. These results identify a certain age-and timedependency of NRF2 effects, which is intriguing. More broadly, our data suggest that specific downstream molecular events may already have taken root, to irrevocably compromise NSPC function, by the end of the critical middle-aged period, thus contributing to a resistance to NRF2-based rejuvenation after this time. Dysfunction of proteasome-dependent proteolysis is also heavily implicated in aging and cell senescence (Leeman et al., 2018;Morimoto & Cuervo, 2009). NRF2 regulates proteasome expression, and its activation has been shown to impede cellular senescence, while inactivation of the pathway recapitulates aging phenotypes (Gabriel et al., 2015;Kubben et al., 2016;Schmidlin et al., 2019). Such mechanisms may be important in determining the age-dependent NRF2 effects seen in our study. Interestingly, other studies have indicated that old mouse NSPCs can be activated through specific intrinsic manipulations (Leeman et al., 2018;Seib et al., 2013). In the context of the current work, since NRF2 has a multitude of targets besides GCLM, it is possible that age-associated differences exist in the ability of NRF2 to activate certain downstream genes versus others due to cell-intrinsic or other extrinsic influences coming from the older niche. For instance, age-related epigenetic alterations in the NRF2 pathway may be involved (Guo et al., 2015). Such processes will need to be further investigated. Nevertheless, our work provides important information regarding specific time-periods during which NSPCs may be more amenable, or resistant, to change-a fundamental subject that needs to be understood but one on which not much is known.
In the larger context of the presented work, we comment that although NRF2 is known as major transcription factor, essential to the cell's survival and homeostatic mechanisms, its specific contribution and importance in stem cells is only recently emerging (Bryan et al., 2013;Schmidlin et al., 2019;Suzuki & Yamamoto, 2017). Stem cells, including pluripotent/embryonic stem cells and adult tissue stem cells, possess unique metabolic programs and reduction-oxidation (or redox) states to sustain proliferation while maintaining pluripotency, multipotency, and/or specified differentiation (Dai et al., 2020). In this vein, it has been shown that NRF2 may govern stem cell function through the modulation of redox and metabolic pathways involving mitochondria and the proteasome (Holmstrom et al., 2016;Jang et al., 2014). In particular, NRF2, by its ability to control cellular reactive oxygen species (ROS) levels, would promote an optimal intracellular redox environment increasingly recognized as critical to stem cell function (Hochmuth et al., 2011;Madhavan, 2015;Noble et al., 2003;Rafalski & Brunet, 2011). Studies by Khacho et al. (2016) suggest that changes in mitochondrial dynamics during neural stem cell development regulate cell fate decisions through a ROSdependent NRF2-mediated transcriptional process (Khacho et al., 2016). The metabolic reprogramming from oxidative phosphorylation to glycolytic energy production seen during the induction of pluripotent stem cell differentiation is also dependent on ROSmediated NRF2 activation (Hawkins et al., 2016;Zhou et al., 2016).
Besides these metabolic and redox effects, NRF2 is also known to directly regulate cell division and phenotypic fate by interacting with other transcription factors and cell cycle regulators involved in maintaining cellular self-renewal, multipotency/pluripotency, and differentiation (Wakabayashi et al., 2015;Zhu et al., 2013). Our work aligns with these studies and highlights NRF2 as a crucial player in aging stem cells.
In conclusion, our study provides evidence that enriching NRF2 expression during a critical time of aging can meaningfully support NSPC activity and function. These data implicate NRF2 as a powerful age-relevant regulator of NSPCs. Understanding the molecular basis of NRF2's effects will reveal fundamental aspects of NSPC biology that underlie its ability to sustain enduring plasticity and lifelong resilience. Moreover, optimizing NRF2 pathway regulation of downstream targets will likely expand the opportunities for clinical translation of NSPCs.

| Animals
Adult male Fisher 344 rats aged 11 mos and 20 mos were obtained from the National Institutes of Health (NIH-NIA). The rats were
Intraperitoneal Bromodeoxyuridine (BrdU) injections were given at about 4 mos post-AAV to label proliferating and migrating NSPCs (Corenblum et al., 2015;Madhavan et al., 2015). BrdU was delivered at a dose of 50 mg/kg/12 h for 3 days, and the animals sacrificed 4 days afterward.

Fine olfactory discrimination behavior
Rats were subjected to behavioral testing via a fine olfactory discrimination task, which is an established measure of the SVZ NSPC function and neurogenesis in vivo (Corenblum et al., 2016;Enwere et al., 2004). As described previously, the task includes an initial training and subsequent testing stages for Discrete and Fine with the same 2-min time limit and 30-s inter-trial interval. This was repeated so that five total trials were conducted.

Challenging beam test
In order to assess striatum-based motor function, rats were tested through a modified beam walking task (Drucker-Colin & Garcia-Hernandez, 1991). Briefly, rats were trained to traverse a set of wooden beams of three different widths (15 mm, 20 mm, and 30 mm) consisting of a start platform at 100 cm above floor height, and an end platform (with rat's home cage) also at 100 cm above floor height. The rats were then evaluated through different measures to analyze motor strength and coordination. Training and testing are performed in the dark during animal's awake cycle. Briefly, training consists of 3 days, where each animal is placed on the beam and allowed to traverse the length in each of three trials. A 30-s rest time is allowed between runs. Testing consisted of the rat being placed on each of the three beams consecutively and allowed to traverse the length. Two trials were completed for each width of beam. If the animal did not cross the beam in 120 s, the trial was considered unsuccessful. Evaluations are based on successful beam crossings, total time to traverse the beam, and foot slip errors. Specifically, quantification of total traverse time was taken, in seconds, and consisted of only trials where an animal successfully crossed the entire beam and did not fall. For an overall task assessment, a composite score was also generated giving each animal a starting score of 3. One point each was subtracted if the animal made a foot slip error or scooted across the beam instead of using its limbs to cross. If the animal fell or did not cross the beam, it was given a score of zero.

| Immunohistochemistry
Sections were blocked [10% normal goat serum, 0.5% triton-X-100 in tris-buffered saline (TBS, pH 7.4)] and incubated in primary antibody overnight at room temperature (RT). Primary antibodies were detected in a 2-h incubation at RT with secondary antibodies coupled to fluorochromes Alexa 488, 594, 647 (Life Technologies-Molecular Probes) and counterstained with 4′,6′-diamidino-2-phenylindole, dihydrochloride (DAPI, Life Technologies). Alternatively, a chromogenic method was used in which primaries were exposed to biotinylated secondary antibodies (Vector Laboratories) followed by treatment with ABC reagent (Vector Laboratories) and 3′-Diaminobenzidine  Table S1].  (Madhavan et al., 2012. Using the optical fractionator probe, BrdU cell counts were conducted through the dorsolateral SVZ in sections at 480 μm intervals across the rostrocaudal axis of the structure. In terms of the dorsoventral extent of the SVZ counted, it covered the SVZ area to a point midway between the genu of the corpus callosum and the anterior commissure crossing.

| Stereology and cell counting
In all cases, after section thickness was determined, guard zones were set at 2 μm each at the top and bottom of the section. All contours were drawn around the region of interest at 2.5x magnification. Clear uniformly labeled nuclei were counted under a 63x oil immersion objective using a grid size of 40 × 40 μm and a counting frame size of 60 × 60 μm. The counting frame was lowered at 1-2 μm interludes and each cell in focus was marked. The Gundersen method for calculating the coefficient of error was used to estimate the accuracy of the optical fractionator results. Coefficients obtained were generally less than 0.10.
Data were expressed as mean ± SEM of the total number of cells obtained across rostral to caudal sections counted in each experimental group.

| Microscopy
Fluorescence analysis was performed using a Zeiss LSM880 confocal microscope (Zeiss). Z sectioning was performed at 1-2 μm intervals in order to verify the co-localization of markers. Image extraction and analysis was conducted via the Zen Blue software (v2.5; Zeiss).
A Zeiss M2 Imager microscope connected to an AxioCam Mrc digital camera connected was used for brightfield microscopy. A Leica DMI 6000 inverted microscope (Leica Microsystems) equipped with Leica Application Suite-Advanced Fluorescence 3.0 and a Hamamatsu Flash 4.0 sCMOS greyscale camera was used to image entire sections using a 5X dry objective. These pictures were captured using the Leica LAS-X version 3.7 software (Leica Microsystems) and used to generate stitched images that showed broader views of the AAV-eGFP transduction.

| Statistical analyses
Sigmaplot 11 and GraphPad prism 8 software were used for statistical analyses. For comparing two groups, t tests were used. For comparisons between three or more groups, one-way analysis of variance (ANOVA) followed by Tukey's or Bonferroni's post hoc test for multiple comparisons between treatment groups was conducted. A two-way repeated measures ANOVA with Tukey's post hoc test was applied to the olfactory behavioral data. Differences were accepted as significant at p < 0.05. Statistical details as pertaining to each experiment are provided within the relevant results and legend sections.

CO N FLI C T O F I NTE R E S T
The authors declare no competing financial interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request. As such, we will follow guidance provided by the journal for sharing the data.