(2024) 21:182
Wahl et al. Journal of Neuroinflammation
https://doi.org/10.1186/s12974-024-03182-9

Journal of Neuroinflammation

Open Access

RESEARCH

Nanoligomers targeting NF‑κB and NLRP3
reduce neuroinflammation and improve
cognitive function with aging and tauopathy
Devin Wahl1,2, Sydney J. Risen3,4, Shelby C. Osburn1,2, Tobias Emge1,2, Sadhana Sharma5, Vincenzo S. Gilberto5,
Anushree Chatterjee5, Prashant Nagpal5, Julie A. Moreno3,4 and Thomas J. LaRocca1,2*

Abstract
Neuroinflammation contributes to impaired cognitive function in brain aging and neurodegenerative disorders
like Alzheimer’s disease, which is characterized by the aggregation of pathological tau. One major driver of both ageand tau-associated neuroinflammation is the NF-κB and NLRP3 signaling axis. However, current treatments targeting NF-κB or NLRP3 may have adverse/systemic effects, and most have not been clinically translatable. In this study,
we tested the efficacy of a novel, nucleic acid therapeutic (Nanoligomer) cocktail specifically targeting both NF-κB
and NLRP3 in the brain for reducing neuroinflammation and improving cognitive function in old (aged 19 months)
wildtype mice, and in rTg4510 tau pathology mice (aged 2 months). We found that 4 weeks of NF-κB/NLRP3-targeting Nanoligomer treatment strongly reduced neuro-inflammatory cytokine profiles in the brain and improved
cognitive-behavioral function in both old and rTg4510 mice. These effects of NF-κB/NLRP3-targeting Nanoligomers
were also associated with reduced glial cell activation and pathology, favorable changes in transcriptome signaturesof glia-associated inflammation (reduced) and neuronal health (increased), and positive systemic effects. Collectively,
our results provide a basis for future translational studies targeting both NF-κB and NLRP3 in the brain, perhaps using
Nanoligomers, to inhibit neuroinflammation and improve cognitive function with aging and neurodegeneration.
Keywords NF-κB, NLRP3, Neuroinflammation, Aging, Tauopathy, Cognitive function
Introduction
Aging is associated with declines in cognitive function,and it is the primary risk factor for most neurodegenerative diseases, including Alzheimer’s disease (AD). These*Correspondence:
Thomas J. LaRocca
tom.larocca@colostate.edu
1
Department of Health and Exercise Science, Colorado State University,
1582 Campus Delivery, Fort Collins, CO 80523, USA
2
Columbine Health Systems Center for Healthy Aging, Colorado State
University, Fort Collins, CO, USA
3
Department of Environmental and Radiological Health Sciences,
Colorado State University, Fort Collins, CO, USA
4
Brain Research Center, Colorado State University, Fort Collins, CO, USA
5
Sachi Bio, Colorado Technology Center, Louisville, CO, USA

declines in cognitive function are closely linked withthe accumulation of pathological tau, a hallmark of AD
that also increases with aging [1, 2]. One key “macromechanism” that contributes to both brain aging andtau pathology is neuroinflammation, which is characterized by innate immune signaling, glial cell activation,
reduced neuronal health and the release of neurotoxic,
pro-inflammatory cytokines [3]. In fact, evidence suggests that neuroinflammation may occur early in brain
aging and both precede and be exacerbated by tauopathy[4]. As such, identifying strategies to reduce neuroinflammation in the context of aging and tauopathy is an important goal [5].
The transcription factor Nuclear Factor Kappa B (NFκB) and inflammasome member NLR Family Pyrin© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to theoriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of thislicence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​
mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Wahl et al. Journal of Neuroinflammation(2024) 21:182

Domain Containing 3 (NLRP3) are key drivers of ageand tau-associated neuroinflammation, and both play
central roles in adverse processes that drive brain aging
and AD (e.g., immune activation and pro-inflammatory
signaling) [6, 7]. The roles of these proteins in brain aging
and neurodegenerative disease are multifaceted, and
NF-κB in particular may also be neuroprotective (e.g.,
by regulating synaptic plasticity and neurotrophic factors) [8]. However, a preponderance of recent studies
have demonstrated that the inhibition of either NF-κB or
NLRP3 alone reduces immune activation/pro-inflammatory cytokine release in the brain and improves cognitive
function [9–11], suggesting that the NF-κB/NLRP3 signaling axis may be a promising therapeutic target. Despite
these findings, current treatments that target NF-κB andNLRP3 may have off-target effects (e.g., by inhibiting
activity of other enzymes) [12] and/or poor biodistribution, and many systemic anti-inflammatory treatments(e.g., immunosuppressive steroids) do not cross the
blood–brain barrier. To address these concerns, our teamrecently developed a novel Nanoligomer (nucleic acidtherapeutic) cocktail that targets both NF-κB and NLRP3Page 2 of 18

DNA, to inhibit transcription, and mRNA, to inhibittranslation (Fig. 1A). Nanoligomers comprise an antisense peptide nucleic acid (PNA) conjugated to a gold
nanoparticle for improved delivery [13–18], and this particular Nanoligomer cocktail was developed via in-depth
screening studies for different targets to reduce neuroinflammation, in which NF-κB emerged as the top single
target and NLRP3 showed a synergistic interaction [16,19]. Our recent reports demonstrate that this NF-κB/
NLRP3 Nanoligomer cocktail is highly specific, crosses
the blood–brain barrier, is bioavailable across various
brain regions [18, 20], downregulates its targets both at
the RNA and protein level, and inhibits neuroinflammation in vitro and in vivo [15, 16, 18–20], yet its influence
on cognitive function in the context of brain aging andtauopathy has not been thoroughly explored.
In the present study, we evaluated the efficacy of targeting both NF-κB and NLRP3 in brain aging and tauopathy by treating both old wildtype mice and transgenic
rTg4510 mice (a model of tauopathy commonly usedin neurodegeneration research) with our Nanoligomer
cocktail. We found that this treatment: (1) improved

Fig. 1 Short-term Nanoligomer treatment downregulates NF-κB and NLRP3 in the brain. A NF-κB and NLRP3 interact to drive inflammation,
but Nanoligomers that block their translation may reduce age- and tau-associated neuroinflammation. B–E Western blot data showing protein
levels of NF-κB, phosphorylated (p)-NF-κB, NLRP3 and Cluster of Differentiation 68 (CD68) in the hippocampus of young (8 months) and older(19 months) wildtype mice, and older mice 16 h after treatment with a NF-κB/NLRP3-targeting Nanoligomer cocktail. Raw immunoblots shownat bottom left and full blots shown in supplementary data file. Data analyzed via ordinary one-way ANOVA and Tukey’s post-hoc testing (p-NF-κB,
F (2, 9) = 1.53; NLRP3, F (2, 9) = 6.89) or non-parametric one-way ANOVA for with Dunn’s post-hoc testing for non-normally distributed data (NF-κB,
Kruskal–Wallis statistic = 7.47; CD68, Kruskal–Wallis statistic = 5.11). N = 4/group for all analyses; *p < 0.05

Wahl et al. Journal of Neuroinflammation(2024) 21:182

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Fig. 2 Long-term treatment with Nanoligomers targeting NF-κB and NLRP3 improves cognitive-behavioral function in old wildtype and tauopathy
mice. A Novel Object Recognition index (short-term recognition memory) quantified as time exploring the novel object versus total object
exploration time in young, old and old Nanoligomer-treated wildtype mice, as well as littermate (LM) controls, rTg4510 tauopathy and rTg4510
Nanoligomer-treated mice. B Time spent in open arms during Elevated Plus Maze testing, a reflection of anxiety-like behavior, in the same mice.
Data analyzed via one-way ANOVA and group differences determined via Tukey’s post-hoc multiple comparison test. Novel Object Recognition:
young and old mice, F (2, 24) = 7.61; rTg4510 mice, F (2, 15) = 6.03. Elevated plus maze: young and old mice, F (2, 24) = 4.31; rTg4510 mice, F (2,15) = 2.97. N = 6–11/group, all data points shown; *p < 0.05

cognitive function and broadly reduced neuroinflammation in both models; (2) reduced tau pathology andglial cell activation while conferring neuroprotection; (3)reversed transcriptome changes common to brain aging
and tauopathy; and (4) exerted favorable systemic effects.
Our results show that selective targeting of both NF-κB
and NLRP3 is a potent strategy for reducing neuroinflammation, and they provide a basis for similar studies
in larger animal models and/or pilot clinical trials.

Results
Short‑term Nanoligomer treatment downregulates NF‑κB
and NLRP3 in the hippocampus

NF-κB and NLRP3 are central mediators of age-related
inflammation (i.e., “inflammaging”), neuroinflammation,
innate immune responses and cytokine release, including in the brain [6, 7, 21, 22]. Our published data showthat NF-κB/NLRP3-targeting Nanoligomers downregulate these targets in multiple brain regions [18, 20]. However, to confirm this in the present study, we first treatedolder (aged 19 months) male and female C57Bl/6J micewith the same Nanoligomer cocktail or saline control(1 × intraperitoneal injection; 150 mg/kg body weight).
Younger (aged 8 months) male and female mice, as areference group, also received a saline injection. After
16 h, mice were euthanized and immunoblots were performed on isolated whole hippocampus. We observed
significant reductions in both NF-κB and NLRP3 in oldtreated vs. untreated mice (Fig. 1B, D), and a modest but

non-significant increase in phosphorylated (activated)
NF-κB in older vs. young control animals that was absent
in older treated animals (Fig. 1C). These effects of acute
Nanoligomer administration on central mediators of neuroinflammation and innate immune responses are consistent with our reports showing that the drug crosses the
blood–brain barrier and engages its targets [13, 14, 16,
18, 20], suggesting the potential for protective effects on
mediators of neuroinflammation downstream of NF-κB/
NLRP3 (e.g., reactive glia, pro-inflammatory gene induction and cytokine release) [23, 24]. Consistent with thisidea, even with the short duration of this treatment, we
also found a nearly significant trend for reduced expression of Cluster of Differentiation 68 (CD68), a marker ofpro-inflammatory microglia in Nanoligomer-treated old
mice (Fig. 1E).
Long‑term Nanoligomer treatment improves cognitive
function in old wildtype and tauopathy mice

Although NF-κB and NLRP3 play complex roles in brain
health [8], most data suggest that inhibiting these proteins individually improves cognitive function [25, 26].
Therefore, given our finding that acute Nanoligomer
treatment reduced NF-κB and NLRP3 in the hippocampus (a key area of the brain involved in memory andcognitive function) and the central role of neuroinflammation in cognitive dysfunction with brain aging and AD[27], we next tested the hypothesis that long-term Nanoligomer treatment (150 mg/kg body weight; 3 × week forWahl et al. Journal of Neuroinflammation(2024) 21:182

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Fig. 3 Broad reductions in neuroinflammation with Nanoligomer treatment are related to greater cognitive-behavioral function in old wildtype
and tauopathy mice. A Principal Component 1 (PC1) from PC analysis of 36 cytokines in hippocampus of young, old and old Nanoligomer-treatedwildtype mice (left panel). Correlation of PC1 values with Novel Object Recognition Index scores in the same mice (middle panel) and heatmap
showing magnitude of individual cytokine correlations with Recognition Index (right panel). B The same PC and cytokine analyses in littermate(LM) controls, rTg4510 tauopathy and rTg4510 Nanoligomer-treated mice. PC group comparisons analyzed via one-way ANOVA and Tukey’s
post-hoc multiple comparison test (young and old mice, F (2, 19) = 15.64; rTg4510 mice, F (2, 15) = 4.05). Correlations determined as simple Pearson
correlation coefficients (r), and all analyses N = 6–11/group; *p < 0.05

one month) would improve cognitive function in old
C57Bl/6J male and female mice. In addition, because
tauopathy (a key hallmark of AD) is associated with
NF-κB/NLRP3 activation [28] and cognitive dysfunction [29], we also treated 2 month-old male and female
rTg4510 mice, a common model of tauopathy based on
expression of human P301L tau in the forebrain, using
the same approach. Importantly, we did not note any
adverse effects with this treatment, and all body weights
remained stable. After the treatment, we measured shortterm recognition memory via Novel Object Recognition
testing and anxiety-like behavior via Elevated Plus Mazetesting. In Novel Object Recognition testing, we foundthat treated older and tauopathy mice performed significantly better (as measured by recognition indices) thanuntreated controls, and similarly to younger mice and littermate controls, respectively (Fig. 2A). In the ElevatedPlus Maze test, we observed a non-significant trend fortreated older and tauopathy mice to spend more time inopen maze arms than untreated mice, reflecting reduced
anxiety (Fig. 2B). Finally, in tests of grip strength, which
we measured because of its relationship to cognitive

function and overall health [30], we found a slight but
non-significant trend for improvements in old treatedmice, although these effects were not noted in treatedtauopathy mice, and among all animals we observed nodifferences in frailty, which is closely associated with cognitive decline [31] (Supplementary Figure S1). Future,
more comprehensive studies are needed to assess how
NF-κB/NLRP3-targeting Nanoligomer treatment affects
other cognitive and peripheral functional outcomes (e.g.,
spatial memory, treadmill endurance, wire hang, rotarod
performance). However, these results show that longterm Nanoligomer treatment improves several domainsof cognitive-behavioral function in old, non-transgenic
mice, and in tauopathy model mice, without overt
adverse effects on neuromuscular function/health.
Broad Nanoligomer‑induced reductions
in neuroinflammation correlate with cognitive function

The activation of NF-κB and NLRP3 via aging or
tauopathy induces the production of many pro-inflammatory cytokines [32]. Therefore, we performed multiplex ELISAs on brains from the same mice (long-term

Wahl et al. Journal of Neuroinflammation(2024) 21:182

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Fig. 4 Reductions in pathological tau in the hippocampus with Nanoligomer treatment. A Quantification and representative
immunohistochemistry images of phosphorylated tau (p-tau T181 and p-tau T231) in hippocampal and frontal cortex sections from littermate(LM) controls, rTg4510 tauopathy and rTg4510 Nanoligomer-treated mice. Insets in lower right corner of images are 2 × magnificationof representative cell bodies indicated in smaller boxes. Note reduction in p-tau staining density with Nanoligomer treatment. B Raw immunoblots
and quantification of total and phosphorylated tau species (p-tau T205 and p-tau S396) in the same mice (full blots shown in supplementary
data file). Data analyzed via one-way ANOVA and group differences determined via Tukey’s post-hoc multiple comparison test (p-tau T181, F(2, 6) = 13.79; p-tau T231 hippocampus, F (2, 6) = 45.62; p-tau T231 cortex, F (2, 6) = 37.18; p-tau S396, F (2, 9) = 3.89; total tau, (F (2, 9) = 21.83).
Immunohistochemistry, N = 3/group; immunoblots, N = 4/group; *p < 0.05

Nanoligomer-treated) described above, to assess levels of key cytokines involved in neuroinflammationand innate immune responses. Principal component
analyses (PCA) revealed that aging and tauopathy
were associated with robust and broad increases inmany cytokines, whereas cytokine levels in treated old
and tauopathy animals were similar to those in young
mice and littermate controls, respectively (Fig. 3A, B,
left panels). At the individual cytokine level, several
important pro-inflammatory and immune-activatingcytokines that increased with aging and tauopathy
included Interleukin 5 (IL-5) and Macrophage-Inflammatory Protein 2 (MIP-2α), but these effects werereduced with Nanoligomer treatment. Similar trends
were noted for most Interleukins (ILs), Monocyte Chemoattractant Proteins (MCPs), and Interferons (IFNs)(Supplementary Figure S2). Additionally, we foundthat cytokine levels (global/PC-based and individual)

were significantly inversely correlated with shortterm recognition memory (Fig. 3A, B center and rightpanels), which is consistent with existing evidence
that increased cytokine levels in the brain are associated with cognitive decline [33]. Together, these data
show that long-term NF-κB/NLRP3-targeting Nanoligomer treatment broadly reduces neuroinflammation,
including many cytokines that are directly modulated
by NF-κB and NLRP3 [22], and that these changes may
contribute to improvements in cognitive function in
old wildtype and tauopathy model mice.
Reductions in pathological tau with Nanoligomer
treatment

Importantly, tau pathology can both activate and be exacerbated by NF-κB and NLRP3 [34–36], driving cycles
of inflammation-mediated tau propagation and toxicity [37], and phosphorylated (activated) tau is closely

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Fig. 5 Changes in glial cell and neuronal numbers with Nanoligomer treatment in tauopathy mice. A Representative immunohistochemistry
images (left) and counts (right) for cells positive for Fox-3 (NeuN) neuronal marker in CA1 and CA2 regions of the hippocampus in littermate (LM)controls, rTg4510 tauopathy and rTg4510 Nanoligomer-treated mice. B Immunohistochemistry images (left) and counts (right) for cells positive
for Ionized Calcium-Binding Adapter molecule 1 (IBA1) and Glial Fibrillary Acidic Protein (GFAP) in the same mice. Data analyzed via one-way ANOVA
and group differences determined via Tukey’s post-hoc multiple comparison test (CA1 NeuN, F (2, 6) = 57.69; CA2 NeuN, F (2, 6) = 27.59; IBA1, F (2,6) = 5.95; GFAP, F (2, 6) = 15.16). N = 3/group; *p < 0.05

associated with cognitive dysfunction [38]. To characterize the effects of Nanoligomer treatment on tau, we
performed immunohistochemistry for two tau residues(phosphorylated at threonine-181 and threonine-231)
that have been associated with AD pathology and progression [39, 40]. We found that these tau species
increased in hippocampus and cortex of rTg4510 mice,similar to what others have reported in tauopathy models[41, 42], and that this effect was attenuated with Nanoligomer treatment (Fig. 4A). We also performed immunoblots for tau, including for phosphorylated species
more typical of later pathology, and we found that longterm Nanoligomer treatment was associated with modest
reductions (p = 0.05) in both total and phosphorylated (atserine 396) tau using this approach (Fig. 4B).
Finally, in these same mice, additional immunohistochemistry analyses showed that Nanoligomer treatment
and the reduced tau burden we observed were associated with: (1) neuroprotection, as reflected by greaternumbers of Fox-3 (NeuN)-positive cells in the CA1 andCA2 regions of the hippocampus (Fig. 5A); and (2) fewer

potentially reactive glia, as reflected by reductions in
Glial Fibrillary Acidic Protein (GFAP)-positive astrocytes
and a non-significant trend for reductions in Ionized Calcium-Binding Adapter molecule 1 (IBA1)-positive microglia (Fig. 5B). Although GFAP and IBA1 counts do notreflect neuroinflammation per se, these results suggestthat NF-κB/NLRP3-targeting Nanoligomer treatment
may suppress tau pathology and confer neuroprotection,and that this effect may involve changes in glial cell-associated processes.
Reversal of age‑related glial cell activation
with Nanoligomer treatment

Although aged wildtype mice do not develop tau pathology, they do develop gliosis like humans. Indeed, inhuman aging/AD, glial cells (astrocytes and microglia)
become immune-activated, secreting cytokines that
impair neuronal and cognitive function [43]. This neuroinflammation is also associated with morphologicalchanges [44, 45], including alterations in process length[46] and branching [47, 48], which have detrimental

Wahl et al. Journal of Neuroinflammation(2024) 21:182

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Fig. 6 Reversal of age-related cellular changes in astrocytes and microglia with Nanoligomer treatment. A Immunofluorescence images of Glial
Fibrillary Acidic Protein (GFAP) staining in hippocampus sections from young, old and old Nanoligomer-treated wildtype mice. Note generalincrease in GFAP/altered astrocyte morphology in old mice that is reversed with Nanoligomer treatment (scale bar 50 µm). B Quantificationof GFAP + cells and process length and branch number. C Immunofluorescence images of Ionized Calcium-Binding Adapter molecule 1 (IBA1)
staining in the same mice. Note general increase in IBA1/altered microglial morphology in old mice that is reversed with treatment (scale bar 50µm). D Quantification of IBA1 + cells and process length and branch number. E Immunohistochemistry counts for Fox-3 (NeuN)-positive cells(neurons) in CA1 and CA2 regions of the hippocampus in the same mice. Data analyzed via one-way ANOVA and group differences determined
via Tukey’s post-hoc multiple comparison test (GFAP, F (2, 8) = 11.64; IBA1, F (2, 16) = 22.10; CA1 NeuN, F (2, 6) = 0.20; CA2 NeuN, F (2, 6) = 9.81).
*p < 0.05 vs. young; #p < 0.01 vs. old control

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Fig. 7 Transcriptome signatures of inflammation and reduced neuronal health with aging and tauopathy are reversed with Nanoligomer
treatment. A Heatmaps showing Log2Fold differences for the top 1000 increased and decreased transcripts in old vs. young control miceand rTg4510 tauopathy mice vs. littermate (LM) controls (top heatmap rows), and Log2Fold differences for the same genes/transcripts in treated old
and rTg4510 mice vs. their untreated counterparts (bottom heatmap rows). Venn diagrams in center show genes/transcripts that were increased
or decreased in both old and rTg4510 mice, and genes/transcripts that were reversed in terms of relative expression in both treated old and treated
rTg4510 mice. B Gene ontology analyses of genes/transcripts that were increased or decreased in both old and rTg4510 mice. C Gene ontology
analyses of genes/transcripts that were increased or decreased in both treated old and treated rTg4510 mice. D Heatmaps showing hierarchicalclustering and correlations between gene/transcript expression levels in bulk RNA-seq data from the present study and a single-cell RNA-seq
dataset. Cell types associated with major gene expression clusters annotated; note glial cells associated with increased clusters and neurons
associated with decreased clusters in both old and rTg4510 mice, and partial reversal of these patterns in heatmaps showing Nanoligomer
treatment. E Quantifications and raw immunoblots for Cluster of Differentiation 68 (CD68) and Brain-Derived Neurotrophic Factor (BDNF) in animals
from all groups (full blots shown in supplementary data file). Gene expression differences analyzed using DESeq2 algorithms and immunoblot data
analyzed via one-way ANOVA with group differences determined via Tukey’s post-hoc multiple comparison test (CD68: young and old mice, F (2,9) = 2.366; CD68, rTg4510 mice, F (2, 9) = 11.25; BDNF: young and old mice, F (2, 9) = 4.74; rTg4510 mice, F (2, 9) = 0.71). N = 4/group for all analyses;*p < 0.05

Wahl et al. Journal of Neuroinflammation(2024) 21:182

effects on neurons (e.g., synaptic dysfunction). To evaluate the effects of NF-κB/NLRP3-targeting Nanoligomer
treatment on astrocyte and microglia activation/morphology with aging, we performed immunofluorescencestaining on cortex tissue from old, old treated, and young
mice. Consistent with previous findings showing thatastrocyte activation increases with aging [44, 49], wefound evidence of age-related increases in GFAP [50],but this effect was less pronounced in old treated mice
(Fig. 6A). Similar to previous studies [51, 52], we also
found that aging was associated with reduced astrocyte
process length and branch number (Fig. 6B). However,these effects were reversed with Nanoligomer treatment. We also found age-related increases in the activation of microglia, as reflected by increases in IBA1
(Fig. 6C). This effect of aging on microglia was notaltered by Nanoligomer treatment, although we didobserve changes in microglial process length and branch

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number in old animals that were attenuated with treatment (Fig. 6D). Finally, similar to our observations in
rTg4510 mice, using immunohistochemistry we foundthat all of these glial cell changes were associated with areduction in NeuN + cells in old mice that was absent intreated animals, although only in the CA2 region of the
hippocampus (Fig. 6E). Collectively, these results suggest that the beneficial effects of Nanoligomers targeting
NF-κB/NLRP3 in old mice may be due in part to reducing age-related changes in glial cells and the preservationof neurons in the hippocampus.
Transcriptome signatures of inflammation and reduced
neuronal health are reversed with Nanoligomer treatment

Perturbations in the transcriptome affect downstream
biological processes that mediate brain aging/AD [53].
To provide broad insight into how Nanoligomer treatment affected genes/pathways most related to aging and

Fig. 8 Favorable peripheral/systemic changes with long-term Nanoligomer treatment. A Cytokine concentrations in liver of young, old and oldNanoligomer-treated wildtype mice. N = 4/group; *p < 0.05 vs. young; #p < 0.05 vs. old. B Representative immuno-histochemistry images showing
infiltrating immune cells at portal vein border (white arrows) and C quantification of immune cell infiltration in the same mice. *p < 0.01. D Plasma
levels of fibroblast growth factor 21 (FGF-21) measured by ELISA in the same mice. Data analyzed via one-way ANOVA with group differences
determined via Tukey’s post-hoc multiple comparison test. Liver ELISAs: F (2, 23) = 1.44, 7.80, 5.54, 1.98, 2.00, 18.35, 29.98, 2.46 and 12.00; pathology
scores: F (2, 6) = 72.57; plasma ELISA: F (2, 23) = 3.08. N = 6–11/group; *p < 0.01

Wahl et al. Journal of Neuroinflammation(2024) 21:182

tauopathy, we performed RNA-seq on the hippocampus.
We found that aging and tauopathy were associated withgene expression changes (Fig. 7A, top rows of heatmaps)that were largely reversed in terms of relative expression with Nanoligomer treatment (Fig. 7A, bottom rowsof heatmaps). For example, one of the top transcriptsthat increased with aging but decreased with Nanoligomer treatment was S100 calcium-binding protein A8(S100A8), which activates NF-κB and induces microglial
activation [54]. One of the top transcripts that increased
with tauopathy but decreased with Nanoligomer treatment was Interleukin 17 Receptor E (IL17RE), which isalso associated with the activation of NF-κB [55] andcognitive dysfunction [56].
Given the above observations and because aging andtau pathophysiology intersect [57], to understand how
Nanoligomer treatment affected genes/pathways common to both aging and tauopathy, we compared the top
1000 most increased/decreased transcripts common toboth aging and tauopathy, and those same transcripts
in old and tauopathy-treated mice (Fig. 7A, top Venn
diagrams). Gene ontology (GO) analyses revealed that
increased genes common to both aging and tauopathy reflected pathways associated with inflammation,
immune responses and cognitive dysfunction, including immune receptor activity, major histocompatibility complex (MHC) binding, and synapse pruning(Fig. 7B, left). Decreased genes/transcripts common toboth aging and tauopathy comprised pathways associated with neurotransmitter receptor activity andsignal transduction (Fig. 7B, right). However, thesepatterns were generally reversed with treatment. Thatis, among the transcripts that changed in both treated
old and treated tauopathy mice (Fig. 7A, bottom Venn
diagrams), those that were increased reflected pathways involved in brain aging and neuronal/cognitive
function including ion channel activity, neuronal signaling, and neurogenesis (Fig. 7C, left), whereas common decreased transcripts with treatment in aged andtauopathy mice reflected pathways associated with
inflammation and immune responses, including celladhesion and stress response (Fig. 7C, right).
To determine which cell types might have contributed most to the observed transcriptome patterns,
we performed a deconvolution analysis (intersecting
bulk RNA-seq data with single-cell RNA-seq data toidentify cell types most associated with gene expression differences in the bulk data [58]). This revealed
a common pattern in both mouse/treatment models(Fig. 7D). With aging and tauopathy, increased gene
expression signatures (i.e., of inflammation) werelargely associated with glia, whereas decreased gene
expression signatures were primarily associated with

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neurons—and in all cases these cell type-specific contributions to gene expression were at least somewhat
reversed with Nanoligomer treatment. To confirm ourRNA-seq data, we performed immunoblotting for several proteins whose gene expression levels changed(Figure S4), and we found similar patterns of protein
expression for these targets. Also, given our findings
above suggesting changes in glial cells and neuroprotection, we immunoblotted for CD68 (a marker ofactivated/reactive microglia [59]) and Brain-Derived
Neurotrophic Factor (BDNF), a protein responsible
for maintaining neuronal survival and growth [60](Fig. 7E). We observed reduced CD68 in Nanoligomertreated animals, although this effect was not significant in old mice, but no treatment-associated changes
in BDNF in either model. Although not definitive,these results suggest that Nanoligomer targeting of
NF-κB/NLRP3 favorably influences several importantbiological processes associated with brain aging andtauopathy, particularly those involved in neuroinflammation/immune activation and neuronal health, andthat these effects may be mostly driven by changes inglial cells, upstream of neuroprotection (i.e., because
the inflammation-related gene expression patterns thatchanged with treatment mapped to these cell types indeconvolution analyses, and neuron-intrinsic protective proteins like BDNF were unchanged).
Favorable peripheral changes in response to Nanoligomer
treatment

Finally, current therapies meant to target neuroinflammation in the brain often have off-target effects [12, 61],and drug-induced hepatotoxicity is common following
exposure to many bioactive compounds [62]. The liver
is a key metabolic organ that processes such compounds
and therefore a relevant target for therapies meant tohelp the brain [63]. To understand how Nanoligomer
treatment affects the liver, we performed multiplex ELISAs on liver tissue from young, old, and old treated mice
(Fig. 8A). We found that several pro-inflammatory andimmune-activating cytokines that increased with aging
were reduced with Nanoligomer treatment, including
IFN-Ɣ inducible protein (IP-10) and chemokine ligand 1(CXCL1). Additionally, the anti-inflammatory cytokine
interleukin 10 (IL-10) was reduced with aging, but thiseffect was reversed with Nanoligomer treatment. We also
found that aging was associated with evidence of hepatocyte polyploidy and polymorphonuclear leukocytes in theliver, a key source of tissue damage due to inflammation[64], and these effects were attenuated with Nanoligomer
treatment (Fig. 8B, C). Furthermore, in Nanoligomertreated old mice, these favorable changes in the liver wereassociated with a preservation of circulating (plasma)Wahl et al. Journal of Neuroinflammation(2024) 21:182

levels of fibroblast growth factor 21 (FGF-21), a hormone synthesized by the liver and linked with longevity
and neuroprotection [65, 66]. Collectively, these results
suggest that, in addition to their protective effects in thebrain, Nanoligomers targeting NF-κB/NLRP3 are nothepatotoxic and may even favorably modulate several
markers of inflammation and liver/peripheral health inold mice.

Discussion
Neuroinflammation impairs cognitive function and isa major mechanism of brain aging and AD [5], both ofwhich are linked with tau pathology. As such, there isan urgent need to identify safe and effective treatmentsthat target neuroinflammation [67]. Age- and tau-related
neuroinflammation is characterized by immune activation, glial cell (microglia and astrocytes) reactivity, andthe release of neurotoxic, pro-inflammatory cytokines[68, 69], and many of these events involve signaling via
NF-κB and NLRP3. These two proteins are upstream
mediators of immune activation and key components
of a “central axis” of neuroinflammation [7, 22, 70]. For
example, NF-κB regulates many inflammatory processes,
including the activation of pro-inflammatory cytokines
and the transcription of neuroinflammatory genes [22],and NLRP3 is a major component of the inflammasomethat directly regulates multiple immune response pathways (e.g., complement/cytokine signaling) [71]. Our key
finding is that simultaneous, targeted downregulationof both NF-κB and NLRP3 via Nanoligomers reduces
neuroinflammation and improves cognitive function inmouse models of both aging and tauopathy. This may be
an important, clinically relevant finding, as these small
molecule therapeutics are well positioned for further preclinical and eventual clinical translation.
Several recent investigations have shown that separately targeting either NF-κB or NLRP3 reduces neuroinflammation and improves cognitive function in multiple
mouse models [9, 36, 72, 73]. For example, an NF-κB
peptide inhibitor was shown to reduce inflammatory
cytokine expression and microglial activation in 5xFAD
mice [11], and inhibition of NLRP3 with the small molecule MCC950 is reported to reduce inflammation andimprove cognitive function in APP/PS1 mice [9]. However, although NF-κB and NLRP3 signaling pathways
are interconnected [21], no studies have investigated theeffects of simultaneously targeting both of these proteinsin old and/or tauopathy mice. To investigate this idea
here, we performed an initial proof-of-concept study
and found that short-term treatment with Nanoligomers
targeting NF-κB and NLRP3 caused reductions in target
proteins in the hippocampus of old mice, consistent with
our previous reports on this same cocktail both in vitro

Page 11 of 18[15] and in vivo [18]. Then, to provide translational relevance to this finding, we performed a longer intervention (proportional to that which might be conducted inolder adults), in which we treated both old and tauopathy
model mice with NF-κB/NLRP3-targeting Nanoligomers
for one month. Notably, we found that this intervention
was safe, well-tolerated, and elicited no adverse neuromuscular effects (e.g., on grip strength or frailty) or in theliver, a key metabolic organ (despite the potential protective role of NF-κB in various settings). We also foundthat this treatment improved short-term recognition
memory and reduced anxiety, which is often co-morbidwith dementia [74]. These results are important, because
cognitive decline is a major risk factor for mild cognitive
impairment and AD [75], and our findings are consistent with data from other studies on NF-κB and NLRP3
in mouse models of aging and AD [9, 11]. We note that
intraperitoneal Nanoligomer delivery (the mode of treatment in our study) is a translational limitation, but ourgroup is currently developing alternative approaches foradministering these compounds, including via intranasal
inhalation and oral routes. Demonstrating the efficacy ofsuch approaches will be an important next step in thisline of investigation.
Importantly, numerous pro-inflammatory/cytokine
signaling pathways contribute to neuroinflammationassociated declines in cognitive function [5, 69, 76]. To
address this in the present study, we probed the hippocampus for multiple cytokines that have been linked
with cognitive dysfunction, brain aging and tauopathy.
We found that NF-κB/NLRP3-targeting Nanoligomer
treatment reduced almost all of these cytokines, with
PCA showing a marked reversal of overall cytokine patterns in both aged and tauopathy mice. Moreover, thesecytokine profiles were highly (inversely) correlated withcognitive-behavioral function in all animals studied. Collectively, these results suggest that the mechanism bywhich Nanoligomer treatment improves cognitive function involves broadly reducing neuroinflammation. Thisidea is in agreement with our previous studies showingthat Nanoligomer treatment reduces neuroinflammationand improves cognitive function in prion-disease mice(a model of severe neurodegeneration) [18], and withnumerous other studies showing that targeting neuroinflammation improves cognitive function in mouse models of accelerated brain aging [77]. In future work, it will
be important to identify the specific NF-κB/NLRP3-associated cytokine pathways that impact cognitive function
most directly, as some cytokines (e.g., IL-10) can haveboth protective [78] and deleterious effects in the brain[79].
Beyond specific cytokine pathways, neuroinflammation is linked with protein pathology (including tau

Wahl et al. Journal of Neuroinflammation(2024) 21:182

accumulation) and glial cell dysregulation in aging andAD [4, 80–82]. However, the order of these events is
incompletely understood, and neuro-inflammatory
signaling mediators like NF-κB and NLRP3 have beenreported to both contribute to and be activated bypathology [36]. In our study, we observed several effects
of NF-κB/NLRP3-targeting Nanoligomers that areconsistent with this idea. First, we found that reduced
neuroinflammation with NF-κB/NLRP3-targeting Nanoligomers was associated with reductions in total andphosphorylated tau species representative of both earlyand later aging/AD pathology in rTg4510 mice [39, 40].
Because aggressive, transgene-driven expression of tau
occurs in rTg4510 mice at young ages, our findings may
suggest that the treatment protected against tau-induced
NF-κB/NLRP3 activation that further promotes pathology in these animals. This idea would be in line withother studies showing that tau seeding can be reducedby small molecule inhibitors of NLRP3 [35], and thatpharmacologically targeting neuroinflammation, in general, reduces the burden of phosphorylated tau [83, 84].
Second, in aging wildtype mice, we found that NF-κB/
NLRP3 Nanoligomer-induced inhibition of neuroinflammation was associated with evidence of reduced glial cell
activation. In contrast to rTg4510 and other transgenic/
AD mouse models, aging wildtype mice do not develop
tau or Aβ pathology. They do, however, develop glial cellassociated neuroinflammation, which has been linked
with both age-related cognitive dysfunction and tau
propagation [2, 5, 43, 69, 85]. Thus, our findings could
suggest that NF-κB and NLRP3 also contribute to neuroinflammation regardless of pathology. Indeed, similar
to previous studies showing that astrocytes and microglia become reactive/pro-inflammatory with aging [44,49], here we found morphological and protein expression evidence of increased reactivity in both astrocytes
and microglia of old mice—but NF-κB/NLRP3-targeting
Nanoligomer treatment largely attenuated these effects.
Together with our findings in tauopathy mice, theseobservations may suggest that NF-κB/NLRP3-associatedneuroinflammation is a “proximal” target for enhancing cognitive function with aging and attenuating neurodegeneration (i.e., as depicted in Fig. 1). Future, morecomprehensive studies using NF-κB/NLRP3-targeting
Nanoligomers are needed to confirm the order of these
events, and/or to evaluate the treatment’s effects onother neuroinflammation-associated pathologies (e.g.,Aβ aggregation). Such work may be particularly important given the notable limitations of the rTg4510 mousemodel used here. For example, recent data suggest that
pathology in these animals may be driven in part by
off-target effects of the tau transgene (e.g., disruptionof other genes important to brain function) [86]. In ourPage 12 of 18

data, these genes were modestly differentially expressed(average Log2Fold differences ~ − 0.8) in transgenic animals, but their expression was not significantly alteredwith treatment and/or correlated with cognitive function
metrics. This suggests that our overall findings may nothave been driven by artifacts of the transgenic model butthat the interpretation of our data should, perhaps, betaken with caution and it underscores the need for similar studies in additional transgenic models (e.g., 5xFAD,
APP/PS1 or humanized hTau mice).
Finally, given the protective effects of NF-κB/NLRP3targeting Nanoligomers in both old wildtype and tauopathy mice, along with evidence that aging and tauopathy
may intersect [87], we used transcriptomics (RNA-seq)
to provide insight into conserved mechanisms of action.
We found common transcriptome signatures of aging
and tauopathy reflecting differences in neuronal health,neuroinflammation and immune activation, similar toours and others’ observations in pre-clinical models ofbrain aging [88, 89] and aging/AD in humans [90]. However, these age- and tauopathy-associated transcriptome
effects were largely attenuated with Nanoligomer treatment, and even reversed in some cases. Deconvolution analyses of these data also showed that increased
gene expression patterns with aging and tauopathy were
mostly associated with astrocytes and microglia, whereas
decreased gene expression patterns were mostly associated with neurons. NF-κB/NLRP3-targeting Nanoligomers at least partially reversed these patterns in bothmodels, which is consistent with the idea that the treatment largely affected glial cells (i.e., because the directionof gene expression changes in these cells tracked withthat of inflammatory gene expression). Given the important role of NF-κB/NLRP3 in other cell types, additional
multi-omics and/or perhaps single-cell analyses would be
helpful in confirming these observations and more thoroughly evaluating the up- and down-stream mechanisms
by which targeting NF-κB/NLRP3 protects cognitive
function.

Conclusions and future directions
Our findings demonstrate that Nanoligomers specifically formulated to target NF-κB and NLRP3 are safe
and well-tolerated, engage their target proteins in thebrain, reduce neuroinflammation and improve cognitive function with both aging and tauopathy. Our
molecular/transcriptomic data suggest overlapping
mechanisms of action in these contexts, supporting theidea that NF-κB/NLRP3-associated neuroinflammation
is a key target in aging and neurodegeneration. Futurestudies are needed to determine if this approach will
work in larger animal models and/or humans, and toWahl et al. Journal of Neuroinflammation(2024) 21:182

disentangle differential effects in short-term vs. longterm treatment periods with varying dosages.

Methods
Nanoligomer design and synthesis

Nanoligomers were synthesized by Sachi Bioworks
as previously reported [13–18]. Briefly, Nanoligomers comprise an antisense peptide nucleic acid (PNA)
conjugated to a gold nanoparticle for improved delivery and membrane transport. The PNA sequences,provided in [14, 15], were previously screened for solubility, self-complementarity, and off-targeting in thehuman genome. The PNA portions of the Nanoligomers(NLRP3 Sequence: CTT​C TA​C TG​C TC​ACAGG, NFKB
Sequence: AGT​G GT​ACC​GTC​TGCTA) were synthesized on a Vantage peptide synthesizer (AAPPTec,
LLC) with solid-phase Fmoc chemistry. Fmoc-PNA
monomers were obtained from PolyOrg Inc., with A, C,
and G monomers protected with Bhoc groups. Following synthesis, the peptides were conjugated with gold
nanoparticles and purified via size-exclusion filtration.
Conjugation and concentration of the purified solution
was monitored through measurement of absorbance at
260 nm (for detection of PNA) and 400 nm (for quantification of nanoparticles). The sequence of the synthesized PNA was confirmed using LC–MS [13].
Animal husbandry

Older and younger male and female C57Bl/6J mice (aged19 months and 8 months, respectively) were purchasedfrom the National Institute on Aging aged rodent colony.
Male and female rTg4510 mice and appropriate littermate controls (2 months of age) were purchased from theJackson Laboratory (strain #024854). For the first partof the study (proof of concept to show that the Nanoligomer cocktail reduced both NF-κB and NLRP3 in thebrain), cohorts were treated with Nanoligomer acutely(1 × intraperitoneal injection; 150 mg/kg body weight)
and then studied 16 h later. For long-term treatment,
mice were treated for 1 month (150 mg/kg body weight;
3 × week). Mice were housed throughout all experiments
at ~ 18–23 °C on a reverse 12 light/12 dark cycle. Freshwater and ad-libitum food (Tekland 2918; 18% protein)
was routinely provided to all cages. Animals were consistently health checked by the veterinary staff at Colorado State University.
Animal sacrifice and tissue collection

Mice were culled in a fed state in the late morning.
After deep anesthetizing with isoflurane, ~ 1 ml ofblood was removed via cardiac puncture followed byPage 13 of 18

cervical dislocation. The left hippocampus and a pieceof left cortex tissue were removed, flash-frozen on dry
ice, and stored at − 80 °C until further processing. The
right hippocampus and right cortex were processed(paraffin-embedded) for immunostaining (details
below).
Immunoblotting

Frozen hippocampus samples were lysed in RIPA buffer
containing 100 mM KCl, 40 mM Tris HCl, 10 mM Trisbase, 5 mM M
­ gCl2, 1 mM EDTA, and 1 mM ATP (pH7.5), phoSTOP phosphatase inhibitors and cOmplete
protease inhibitors (ThermoFisher). Sample protein
concentration was determined using a BCA kit (ThermoFisher/Pierce), and 20 μg of protein per sample wasseparated by electrophoresis (Bio-Rad Criterion system), transferred to nitrocellulose membranes (TransBlot Turbo, Bio-Rad), and blocked in TBS-T with 5%
Bovine Serum Albumin (BSA). Primary antibodies werepurchased from ABclonal and included phosphorylated nuclear factor kappa B (p-NF-κB, 1:1000 dilution);
nuclear factor kappa B (NF-κB, 1:1000); NLR Family Pyrin Domain Containing 3 (NLRP3, 1:1000); totaltau (1:1000); p-tau S396 (1:1000); p-tau T205 (1:1000);
Cluster of Differentiation 68 (CD68, 1:1000); Indoleamine 2,3‐dioxygenase 1 (IDO1, 1:1000); Interleukin 17
Receptor E (IL17RE, 1:1000); and Brain-Derived Neurotrophic Factor (BDNF, 1:1000). GAPDH (1:2000) was
purchased from Novus Biologicals. Membranes wereincubated in primary antibody (TBS-T in 5% BSA)
for 24 h at 4°C, followed by HRP-conjugated secondary antibodies (TST-T in 5% milk; 1:5000; Cell Signaling) for 1 h. Membranes were then incubated in ECL,
imaged on a ProteinSimple Fluor Chem imaging system, and quantified with ImageJ.
Cognitive‑behavioral and functional testing

Novel Object Recognition: For NOR testing, mice wereassessed for short-term memory performance using nohabituation phase [91]. Briefly, mice were placed in an
opaque arena (50 cm × 50 cm) with two identical objects(tower of Legos) placed 8 cm from each side wall. Afteran inter-trial interval of two hours, mice were returnedto the arena, but one of the old objects was replacedwith a new object of similar shape and color (cell culture flask filled with multi-colored sand). Trials ended
once 20 s total exploration time was reached, or 10 min
had elapsed. If mice were unable to meet the exploration criteria of 20 s or 10 min, they were excluded fromthe analysis. Recognition index was calculated as novel
object exploration time divided by 20 s [91].

Wahl et al. Journal of Neuroinflammation(2024) 21:182

Elevated Plus Maze: Elevated Plus Maze testing was
performed as previously described [90, 92] and consistedof a cross with four arms (67 cm in length; 2 exposedarms, 2 enclosed arms). Mice were placed in the centerof the maze facing an open arm and allowed to freely
explore for 5 min. The amount of time in open arms (ameasure of anxiety-like behavior) was recorded and analyzed with AnyMaze software.
Grip Strength: The grip strength test was performed
as previously described [93]. Mice were tested using theDST-110 Digital Force Gauge (Maze Engineers). A totalof 5 trials were performed per mouse and the highest andlowest values were discarded for analysis. The remaining
3 force values were averaged for each mouse. Data arepresented as g force/g body weight.
Frailty Testing: Mouse frailty scores were assessedusing an established scale [94]. Briefly, trained investigators blindly scored mice on a 31-point scale, which
included measurements of health in the following areas:
integumentary, physical/musculoskeletal, vestibular/
auditory, ocular/nasal, digestive/urogenital, and respiratory systems. Higher scores indicate worse outcomes,
while lower scores indicate better outcomes.
Multiplexed ELISAs

Brain homogenates from all mice were assessed witha 36-plex Procartaplex cytokine/chemokine panel(ThermoFisher) as previously reported [15, 16], which
included the following cytokines and chemokines: ENA78 (CXCL5), Eotaxin (CCL11), GRO-α (CXCL1), IP-10(CXCL10), MCP-1 (CCL2), MIP-1α (CCL3), MIP-1β(CCL4), MIP-2α (CXCL2), RANTES (CCL5), G-CSF(CSF-3), GM-CSF, IFN-α, IFN-γ, IL-1α, IL-1β, IL-2, IL-3,
IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-13, IL-15/IL15R, IL-17A (CTLA-8), IL-18, IL-22, IL-23, IL-27, IL-28,
IL-31, LIF, MCP-3 (CCL7), M-CSF, and TNF-α. For systemic effects (Fig. 7), separate individual ELISAs wereused. Briefly, 25uL of 10 mg/ml hippocampus, cortex,
liver homogenate, or serum was processed using standard ELISA techniques and analyzed on a Luminex MAGPIX xMAP instrument. Standards for each cytokine/
chemokine were used with 1:4 dilutions (eightfold dilutions) and background, controls and sample concentrations were determined from a standard curve using Five
Parameter Logistic curve fit/quantification.
Immunohistochemistry and immunofluorescence

Brain and liver tissues were fixed in 10% neutral buffered formalin at room temperature for at least 48 h.
Brain samples were processed on a Leica TP1020
Automatic Benchtop Tissue Processor and embedded in paraffin wax (Cancer Diagnostics). For immunohistochemistry analyses of phosphorylated tau,

Page 14 of 18

paraffin-embedded tissue was sectioned at 4 µm,
deparaffinized, rehydrated, sodium citate treated, andblocked in Tris A/2% donkey serum (Jackson ImmunoResearch), then incubated overnight in the following
antibodies: phospho-tau (T181, 1:400 dilution; Invitrogen) and phospho-tau (T231, 1:400; ABClonal). Wash
steps were performed using 2% bovine serum albuminand 2% Triton-X in 1 M TBS, and an ABC HRP peroxidase detection kit (Vector Laboratories) and ImmPACTDAB Substrate Peroxidase (HRP) Kit (Vector Laboratories) were used for a chromogen. Slides were counterstained with hematoxylin (ThermoFisher), secured
with a coverslip in mounting medium and stored atroom temperature until imaging. Whole tissue images
were taken on an Olympus BX53 microscope with an
Olympus DP70 camera using an Olympus UPlanSApo
20 × objective (N.A. = 0.75). Representative images
were taken using an Olympus BX53 microscope with
an Olympus DP70 camera using an Olympus UPlanFL
N 40 × objective (N.A. = 0.75). To quantify and selectthe phosphorylation of tau-positive stained cells the
manual threshold on the Count and Measure function of Olympus CellSens software (v1.18) was used.
For immunofluorescence analyses of GFAP, IBA1 andglial cell process length and branching, sections wereincubated overnight in primary antibodies: IBA1 (1:50
dilution; Abcam) and GFAP (1:100; Dako). Slides were
washed with TBS and incubated for 1 h in the dark with
AlexaFluor 555 or 647 (Invitrogen) secondary antibodies at 1:500 dilution and 2% normal donkey serum, thenwashed and incubated in Hoechst dye (1:2000; ThermoFisher) for 3 min. Slides were mounted with Prolong
Gold Antifade mounting media (ThermoFisher) andstored in the dark at 4 °C prior to imaging. Images were
captured using an Olympus BX63 fluorescence microscope equipped with a motorized stage and Hamamatsu
ORCA-flash 4.0 LT CCD camera using a 40 × Olympus
X-Apochromat air objective (N.A. = 0.80). For astrocyte/microglial process length and number analyses,
four regions between the dentate gyrus and CA1-CA3
region of the hippocampus were imaged at 40×. To
identify/quantify processes and branches, skeletonization of GFAP+ astrocytes and IBA1+ microglia was performed using IMARIS software as previously described[95] to trace and quantify stained filaments. For histology analyses of liver, tissues were sectioned on a ThermoFisher HM 325–2 microtome at 5 μm thickness andmounted on positively charged glass slides (Superfrost
Plus, Cancer Diagnostics). Liver sections were deparaffinized and stained with hematoxylin and eosin (Cancer Diagnostics), and whole tissue images were taken
for analysis using an Olympus DP70 camera and an
Olympus UPlanSApo 20 × objective. Livers were scoredWahl et al. Journal of Neuroinflammation(2024) 21:182

for pathological infiltration of immune cells specifically around the portal triad, and for overall hepatocyte
morphology by a trained animal pathologist.
RNA sequencing and transcriptomic analyses

Transcriptome (RNA-seq) and gene expression analyses
were performed on whole hippocampus via methods previously described [89, 90]. Frozen samples (n = 6/group;
3M and 3F) were transferred into Trizol (ThermoFisher)and homogenized on ice (SP Bel-Art ProCulture MicroTube Homogenizer System; VWR Scientific). RNA was
isolated using an RNA-specific spin column kit (DirectZol, Zymo Research) and treated with DNase to remove
genomic DNA. Poly(A)-selected libraries were generated using magnetic oligo dT beads (ThermoFisher) andIllumina TruSeq kits. Libraries were sequenced on an
Illumina NovaSeq 6000 platform to yield > 40 M 151-bp
paired-end FASTQ reads/sample. Reads were trimmed
and filtered with the fastp program [96], then aligned
to the mm10 Mus musculus genome using the STARaligner [97]. Differential gene expression was analyzed
with DESeq2 [98] and genes/transcripts with BaseMean
read counts of < 10 were filtered and removed for analyses[99, 100]. Differentially expressed genes were analyzed
for gene ontology (GO) enrichment using the Metascape program [101], and heatmaps were constructedwith GraphPad Prism software. Deconvolution analyses of bulk RNA-seq data were performed as previously
described [88] using a published protocol and R script[58]. Briefly, single-cell RNA-seq data on mouse hippocampus (SMART-seq, 2019; 10x-SMART-seq taxonomy, 2020) were downloaded from the Allen Brain Map
portal [102], and cells from the hippocampus, including
astrocytes, endothelial cells, microglia, oligodendrocytes and neurons, were sub-setted for analysis. Normalized DESeq2 gene counts for old vs. young mice, old
Nanoligomer-treated vs. old, rTg4510 vs. LM Controls,
and rTg4510 Nanoligomer-treated vs. rTg4510 comparisons in bulk RNA-seq data were filtered for the top 250
genes by p-value, and then sorted by Log2Fold change.
Sorted gene lists were scaled to the single-cell RNA-seq
dataset, normalized expression of the same genes in the
single-cell dataset was obtained and used for dimensional reduction via principal component analysis andhierarchical clustering/heat mapping to obtain cell-type
specific contributions to differences in the bulk RNA-seq
comparisons, and cell types associated with each cluster
were determined using CellMarker 2.0 [103].
Statistics

One-way ANOVA with Tukey’s post-hoc testing was used
to assess differences among groups in protein/cytokine

Page 15 of 18

levels and measures of cognitive function, as well as glial
cell morphology, and simple linear regression (Pearson
correlations) was used to relate cytokine levels to cognitive function data. A non-parametric ANOVA equivalent(Kruskal–Wallis test) was used for small sample sizes
with non-normally distributed data. All data were analyzed and presented using GraphPad Prism software. Differentially expressed genes were detected using DESeq2,
and genes were sorted by p-value and Log2Fold change
to construct volcano plots and heatmaps, respectively.
The Metascape program was used for GO and TRRUST
analyses [101].

Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s12974-​024-​03182-9.
Additional file 1: Figure S1. Nanoligomers targeting NF-κB and NLRP3
increase grip strength in old wildtype mice and have no adverse effects
on overall physical health/frailty. Left: Forelimb grip strength in young,
old and old Nanoligomer-treated wildtype mice, as well as littermate(LM) controls, rTg4510 tauopathy and rTg4510 Nanoligomer-treatedmice. Right: Frailty Index in the same animals. N = 6–11/group; *p < 0.05.
Figure S2. Nanoligomers targeting NF-κB and NLRP3 reverse age- andtauopathy-related increases in many cytokines in the brain. Multiplex
ELISA analyses of prefrontal cortex tissue from young, old and oldNanoligomer-treated wildtype mice, as well as littermate (LM) controls,
rTg4510 tauopathy and rTg4510 Nanoligomer-treated mice. N = 6–11/
group; *p < 0.05 vs. young/LM; #p < 0.05 vs. old/rTg4510; red lines represent
limit of detection. Figure S3. Nanoligomers targeting NF-κB and NLRP3
modulate glial cell morphology in old mice. Representative images from
Fig. 5 (left in each panel) and corresponding images (right in each panel)
showing skeletonization (white lines) for assessing morphology in A
astrocytes stained for GFAP and B microglia stained for IBA1. Figure S4.
Immunoblotting confirmation of hits in RNA-seq data. Raw immunoblots
and quantifications showing protein levels that track with RNA-seq gene
expression levels for Indoleamine 2,3‐dioxygenase 1 (IDO1) and Interleukin 17 Receptor E (IL17RE), both of which have also been linked with differences in cognitive function, in young, old and old Nanoligomer-treatedwildtype mice, as well as littermate (LM) controls, rTg4510 tauopathy andrTg4510 Nanoligomer-treated mice. *p < 0.05; **p < 0.01. Note: same blot/
GAPDH loading controls as in Fig. 7.
Additional file 2.
Author contributions
D.W. designed the study, wrote the paper, generated/analyzed data,
conducted behavioral experiments, and generated/analyzed RNA-seq andimmunoblot data; S.J.R. generated and analyzed immunofluorescence/immunohistochemistry data; S.C.O, T.E., V.S.G., S.S. and A.C. generated and analyzed
cytokine/protein data; P.N. designed the study, provided conceptual insight,
and generated/analyzed data; J.A.M. designed the study and generated/analyzed immunofluorescence/immunohistochemistry data; T.J.L. designed thestudy, wrote the paper, generated/analyzed data, provided conceptual insight,
and provided funding. All authors read and approved the final manuscript.
Funding
This project was supported in part by NIH awards AG078859, AG070562 andAG060302 (T.J.L.) and AG069361 (D.W.), NASA SBIR Awards 80NSSC22CA116,
80NSSC23CA171 and National Academy of Science Health Longevity Catalyst
Award (P.N.), and by intramural (startup) funding from Colorado State University (T.J.L.).

Wahl et al. Journal of Neuroinflammation(2024) 21:182

Data availability
The RNA-seq data generated in this study are available on the Gene Expression Omnibus website under accession number GSE251888. All additional
data are included in the data supplement, and raw data will be made available
upon request to the corresponding author (T.J.L).

Declarations
Ethics approval and consent to participate
This protocol was approved by IACUC #1441 and Laboratory Animal Resources(RRID: SCR_022157) staff at Colorado State University.
Consent for publication
Not applicable.
Competing interests
S.S, V.G, A.C., and P.N. work at Sachi Bio, a for-profit company, where the Nanoligomer technology was developed. A.C. and P.N. are the founders of Sachi
Bio, and P.N. has filed a patent on this technology. All other authors declare nocompeting interest.
Received: 13 February 2024 Accepted: 22 July 2024

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