DRP1 induces neuroinflammation via transcriptional regulation of NF-ĸB.

Domain: biology
Published: 2026-03-16
Editorial outcome: minor_revision

Abstract
Neuroinflammation is a major pathogenic mechanism underlying neurodegenerative diseases.
Understanding how neuroinflammation is regulated is critical to therapeutic development. Here,
we report that dynamin related protein 1 (DRP1), well-recognized for its role in mitochondrial
fission, also functions as a transcription factor that regulates neuroinflammation. Using multiple
inflammatory models, we demonstrate that upon stimulation with pro-inflammatory
lipopolysaccharides (LPS), DRP1 translocates from the cytosol to the nucleus, where it binds to
the promoter region of Rela (encoding NF-κB p65) to activate its gene products and other
downstream inflammatory cytokines. Our data further reveal a significant role of the
proinflammatory lipocalin-2 in the brain. In combination, this study identifies a previously
unrecognized function of DRP1 in mediating neuroinflammation via the NF-κB-lipocalin-2 axis and
highlights DRP1-mediated pathways as potential therapeutic targets for neurodegenerative and
other inflammation-related diseases.

Introduction
Neuroinflammation is a fundamental innate immune response in the central nervous system,
involving the activation of glia cells, the release of inflammatory mediators (such as cytokines and
chemokines), and the generation of reactive oxygen and nitrogen species1. The central role of
chronic inflammation in neurodegenerative diseases such as Alzheimer’s disease (AD), and
Parkinson’s disease (PD) has been well-documented2. Thus, targeting neuroinflammation
represents one of the most promising therapeutic strategies for neurological disorders.
Activation of nuclear factor kappa B (NF-κB) is a central signaling pathway in inflammation
and this family of transcription factors has been intensively investigated as a therapeutic target
for a wide range of diseases3-5. In mammals, the NF-κB family comprises five different members:
p65 (RelA), RelB, c-Rel, p50 (NF-κB1), p52 (NF-κB 2)5, 6. The heterodimer formed by the p65 and
p50 subunits is the most abundant and potent activator of gene transcription6, 7. NF-κB can be
activated by a wide variety of stimuli including bacterial toxins such as lipopolysaccharide (LPS),
oxidative stresses, toxic proteins, and inflammatory cytokines6. Upon activation, NF-κB subunits
assembles into homo- or hetero-dimerized transcription factor complexes which then translocate
into the nucleus and bind to the promoter regions of target genes, leading to the transcription of
pro‑inflammatory cytokines8, 9. To date, NF-κB is known to regulate the expression of hundreds
of different genes8, 9, 3, 10, including LCN211 Therefore, identifying factors that regulate its
expression is highly critical.
LCN2 encodes the lipocalin-2 protein, also known as neutrophil gelatinase-associated
lipocalin (NGAL) and 24p3. Lipocalin-2 is a member of the lipocalin superfamily, a group of
secreted proteins that play diverse physiological roles, including bacteriostasis, cell differentiation,
iron binding, and immune response12, 13. In addition, lipocalin-2 has been reported to act as a
pro‑inflammatory regulator in activated macrophage through NF‑κB signaling14. Although the
LCN2 transcript is widely expressed in many tissues and organs, lipocalin-2 levels are either low
or undetectable in the brain under basal conditions15, 16. However, under pathological conditions
or exposure to inflammatory stimuli, its expression and secretion are increased drastically.
Indeed, elevated levels of LCN2 mRNA and lipocalin-2 have been detected in the post-mortem
brain tissues of PD patients and animal model of PD17. Consistent with its role in
neuroinflammation, accumulating evidence indicates that lipocalin-2 is highly expressed in
reactive astrocytes and microglia18-20. Collectively, these findings suggest that lipocalin-2 is
neurotoxic when upregulated under pathological conditions. However, the mechanism by which
dynamin related protein 1 (DRP1) induces neuroinflammation via the NF-κB-lipocalin-2 axis has
not been elucidated.
DRP1 is a member of the dynamin GTPase superfamily and is best known for its role in
mitochondrial fission21, 22. Independent laboratories have demonstrated that partial inhibition of
DRP1 function is protective in experimental models of neurodegenerative diseases22. We recently
reported that transcript and protein levels of DRP1 are highly elevated in the substantia nigra of
PD patients23, further supporting the potential involvement of this protein in PD pathogenesis.
Consistent with its established role in mitochondrial fission, the protective effects of DRP1
inhibition have been largely attributed to restoring the balance in mitochondrial fission and fusion.
However, additional mechanisms are involved. For example, we recently reported that partial
deletion of DRP1 enhances autophagic flux independent of its role in mitochondrial fission24.
Other investigators have reported that DRP1 predominantly resides in the cytosol, with only ~3%
co-fractionating with mitochondria25. Using live-cell imaging of fluorescently labeled DRP1, a
subsequent study corroborated that much of DRP1 diffusely distributed in the cytoplasm and only
~2.5% of DRP1 puncta engage in mitochondrial fission26. Furthermore, DRP1 has been shown to
colocalizes with other organelles, including lysosomes27, the endoplasmic reticulum28 and
peroxisomes29. In combination, these results indicate that DRP1 is a multifunctional protein that
exerts diverse cellular effects beyond mitochondrial division.
In this study, we provide evidence showing that DRP1 induces neuroinflammation through a
transcriptional mechanism. Using LPS to activate microglia, we observed the translocation of
DRP1 from the cytosol to the nucleus, where it binds to the promoter of the Rela gene, resulting
in the upregulation of its gene products and other downstream inflammatory cytokines, including
Lcn2. Among pro-inflammatory genes, Lcn2 exhibited the highest inducibility and marked cell-
type specificity. Elevated Lcn2 levels were also detected in transgenic mice overexpressing
human α-synuclein. These effects were significantly attenuated in mutant mice with heterozygous
knockout of DRP1. Collectively, our study identifies the DRP1-NF-κB-lipocalin-2 axis as a key
pathway mediating neuroinflammation.

Results
Neuroinflammation in mouse models is reduced by partial DRP1-knockdown
Reduced DRP1 function has been shown to attenuate neuroinflammatory responses in LPS-
treated cultured microglia30, 31. However, the in vivo relevance of these observations and the
mechanism by which DRP1 induces neuroinflammation requires further investigation. To address
these gaps, we used our recently generated Dnm1l+/- mice, which exhibit partial DRP1
deficiency24. Dnm1l+/- mice and their wild-type (WT, Dnm1l+/+) littermates were injected
intraperitoneally with a single dose of LPS (5mg/kg)32, 33, and their ventral midbrain (VMB) tissue
was collected 6h later for RNA analysis. Taking an unbiased approach, we performed NanoString
nCounter gene expression analysis using the Mouse Neuroinflammation Panel, which profiles
757 genes across major inflammatory pathways. Consistent with the proinflammatory effects of
LPS, we observed robust dysregulation of proinflammatory transcripts in WT mice following LPS
administration. In contrast, these responses were significantly attenuated in Dnm1l+/- littermates
(Fig. 1a; Supplementary Data 1). The top 10 differentially expressed genes are shown in Fig. 1b,
with Lcn2 exhibiting the most dramatic induction in LPS-treated WT mice (Fig. 1b). Lcn2 mRNA
levels reached 1510 ± 209.5 counts in Dnm1l+/+ mice, compared to 543.6 ± 199.9 in Dnm1l+/- mice,
a significant attenuation (p = 0.0005; Fig. 1c). No sex difference was detectable, and data were
combined.
To determine whether DRP1 reduction protects against Lcn2 elevation beyond the LPS
model, we crossed Dnm1l+/- mice24 with transgenic mice overexpressing human α-synuclein. At 6
months of age, SNCA+/- mice exhibited a 3.9-fold increase in Lcn2 expression relative to WT
littermates; however, this elevation was fully normalized in double-mutant Dnm1l+/-:SNCA+/- mice
(Fig. 1d). Together, these results demonstrate that partial DRP1 inhibition effectively reduces
neuroinflammatory gene expression, particularly Lcn2.
Reduced proinflammatory gene expression in LPS-treated Dnm1l+/- mice
To validate the NanoString data and determine whether changes in Lcn2 expression correlate
with alterations in other proinflammatory genes, we quantified mRNA levels of selected
inflammatory markers in the VMB of LPS-treated mice. These genes included those known to be
regulated by Lcn214, such as Nlrp3, Il1b, as well as genes reported to regulate Lcn2 expression34,
35, including Rela, Il6, Tnfα. All these transcripts were significantly upregulated in LPS-treated
Dnm1l+/+ mice, but their induction was markedly attenuated in Dnm1l+/- littermates (Fig. 2a),
mirroring the pattern observed for Lcn2 (Fig. 1a-c).
To corroborate the gene expression data obtained from the NanoString and qPCR analysis,
we next quantified proinflammatory protein levels using MSD multiplex immunoassay in VMB
lysates from LPS-treated Dnm1l+/+ and Dnm1l+/- mice. Consistent with the mRNA data, LPS
robustly increased the abundance of several inflammatory proteins, including lipocalin-2, in
Dnm1l+/+ mice, whereas these increases were substantially attenuated in Dnm1l+/- mice (Fig. 2b).
The increases in Lcn2, Cxcl10, and Ccl2 were validated at the protein levels. Interestingly,
additional proteins were detectable but not their transcripts. We believe this is due to key
differences between transcript-level detection (NanoString) and protein-level detection (MSD
multiplex ELISA immunoassay). For example, mRNA can be degraded rapidly while the protein
remains stable. NanoString is highly specific, but it has a narrow dynamic range of mRNA
abundance whereas MSD uses electrochemiluminescence and can detect very low femtogram-
levels protein concentrations. Last, there are temporal differences in gene vs protein expression.
Changes in mRNA often occur earlier and transiently whereas proteins may accumulate or persist
longer. Morphologically, microglia in the Dnm1l+/- animals also appeared less activated (more
ramified structures) as quantified using Sholl analysis of IBA-1 immunostaining (Supplementary
Fig. 1).
Nuclear factor erythroid 2–related factor 2 (NRF2) is a transcription factor known for its
antioxidant and cytoprotective functions and has been reported to modulate NF-κB–mediated
inflammatory responses. Loss of NRF2 enhances inflammation by promoting NF-κB activation
and the expression of downstream pro-inflammatory cytokines, whereas pharmacological or
genetic activation of NRF2 suppresses NF-κB–driven inflammation36-38. Collectively, these studies
suggest that NRF2 serves as a negative regulator of NF-κB. To determine whether NRF2
contributes to NF-κB activity in our model, we analyzed our NanoString dataset and found that
Nfe2l2 (the gene encoding NRF2) mRNA levels in the VMB were not significantly changed
following LPS treatment (Supplementary Fig. 2a). We further validated this observation by qPCR,
which similarly showed no alteration in Nfe2l2 expression (Supplementary Fig. 2b). Together,
these results indicate that NRF2 is unlikely to contribute to the neuroinflammatory response
observed in this study.
Expression of proinflammatory genes and cell-type–specific inducibility of Lcn2
Given that gene expression analyses from whole VMB tissue (Figs. 1–2) do not reveal the cellular
sources of Lcn2, and because cell-type–specific expression of Lcn2 in vivo has not been
comprehensively evaluated, we used laser microdissection to isolate individual microglia,
astrocytes, nigral dopaminergic (DA) neurons, and gamma-aminobutyric acid (GABA) neurons
from the substantia nigra of LPS-treated Dnm1l+/+ and Dnm1l+/- mice. As shown in Fig. 3a, Lcn2
was constitutively expressed in all four cell types, with no detectable differences in basal
expression across genotypes or cell populations (Fig. 3b). Following LPS exposure, Lcn2
expression was significantly increased in microglia, astrocytes, and DA neurons of Dnm1l+/+ mice,
but not in GABA neurons (Fig. 3a). Consistent with whole-tissue findings (Fig. 1b), this LPS-
induced upregulation was absent in microglia, astrocytes, and DA neurons microdissected from
Dnm1l+/- mice (Fig. 3a).
To compare Lcn2 inducibility across cell types, Lcn2 expression in each population was
normalized to basal Lcn2 levels in microglia from untreated Dnm1l+/+ mice. These analyses
revealed that microglia and astrocytes exhibit the highest inducibility of Lcn2 following LPS
stimulation, whereas DA neurons showed a more modest response and GABA neurons showed
none (Fig. 3b). Overall, these data suggest that the increases in Lcn2 observed in whole VMB
tissue (Fig. 1) arise primarily from glial populations, particularly microglia and astrocytes, and to
a lesser extent from DA neurons.
To complement the results obtained from the microdissected cells in the global heterozygous
DRP1-deficient (Dnm1l+/-) model (Figs. 3a-b), we next examined mice with cell-type–specific
conditional deletion of Dnm1l. Our recently generated floxed-Dnm1l mice were crossed with
Cx3Cr1CreERT2 or Aldh1L1CreETRT2 lines to generate microglia-specific (“Microglia-KO”) or astrocyte-
specific (“Astrocyte-KO”) Dnm1l knockdown mice, respectively. Tamoxifen-treated animals were
analyzed 3 weeks later. Laser microdissection followed by Smart-seq2/qPCR confirmed a 32%
reduction of Dnm1l in microglia of Microglia-KO mice and a 44% reduction in astrocytes of
Astrocyte-KO mice (Supplementary Fig. 3). Importantly, Dnm1l levels were unchanged in non-
targeted cell populations, demonstrating successful cell-type specificity.
Consistent with observations from the global Dnm1l+/- mice, LPS significantly increased
expression of proinflammatory genes, including Lcn2, Il6, Il1b, and Tnfα, in microglia and
astrocytes from control floxed (Dnm1l-Loxp+/-) mice, relative to untreated littermates (Fig. 3c–d).
However, this induction was abolished in Microglia-KO and Astrocyte-KO mice (Fig. 3c–d),
indicating that partial loss of Dnm1l in either microglia or astrocytes is sufficient to blunt LPS-
induced inflammatory gene expression. Together, these results demonstrate that both microglial
and astrocytic DRP1 contribute significantly to the neuroinflammatory response induced by LPS,
and that reducing DRP1 in either cell type effectively limits the upregulation of key inflammatory
mediators such as Lcn2.
DRP1 inhibition prevents neuroinflammation via the NF-ĸB pathway
Based on our results showing that partial DRP1 deficiency protects against LPS-induced
neuroinflammation, and considering DRP1’s well-established role in mitochondrial function, we
first examined whether this protection might stem from preventing LPS-induced mitochondrial
dysfunction. To test this possibility, we isolated microglia from adult mice treated with LPS and
measured mitochondrial respiration. No mitochondrial impairment was detected under these
conditions (Supplementary Fig. 4a–b). These results indicate that the reduced neuroinflammatory
response observed in Dnm1l+/- mice (Figs. 1–3) is unlikely to be mediated through preservation
of mitochondrial function.
To further investigate how DRP1 inhibition reduces LPS-induced neuroinflammation and
microglial activation, we performed a chromatin immunoprecipitation (ChIP) assay in primary
microglia treated with LPS for 6 h, the same time point used in our in vivo studies. Because Lcn2
has been reported to be transcriptionally regulated by NF-κB14, and its promoter contains a κB
binding motif (Fig. 4a), we examined whether NF-κB p65 is recruited to the Lcn2 promoter
following LPS exposure. In WT microglia, LPS robustly increased p65 binding to the Lcn2
promoter (Fig. 4b). Strikingly, this enrichment was significantly reduced in microglia from Dnm1l+/-
mice (Fig. 4b), indicating that DRP1 reduction reduces NF-κB recruitment to at least one of its
key proinflammatory targets.
To further test the involvement of NF-κB, we measured proinflammatory cytokines in the
conditioned media of LPS-treated microglia in the presence or absence of the NF-κB inhibitor
Bay11-708239. Pharmacological inhibition of NF-κB markedly reduced cytokine release in WT
microglia (Fig. 4c). Notably, microglia from Dnm1l+/- mice displayed a similarly reduced
inflammatory response (Fig. 4c). These in vitro observations are consistent with our in vivo
findings showing decreased proinflammatory protein levels in the VMB of LPS-treated Dnm1l+/-
mice (Fig. 2b). Together, these results reinforce a model in which DRP1 promotes
neuroinflammation through activation of the NF-κB pathway.
To assess whether mitochondrial alterations contribute to LPS-induced responses in primary
microglia, we performed time-course studies. At the 6h time point, the stage at which
neuroinflammation is clearly induced, WT microglia exhibited neither mitochondrial fragmentation
(Supplementary Fig. 4c–d) nor mitochondrial dysfunction (Supplementary Fig. 4e–h). Even at
later time points (24 h), mitochondrial respiration remained unaffected. At an earlier time point (2
h), LPS transiently induced shorter mitochondria in both Dnm1l+/+ and Dnm1l+/- microglia, but this
morphological change resolved without detectable functional deficits. Collectively, these results
demonstrate three key points: First, LPS activates microglia without inducing mitochondrial
dysfunction under the experimental conditions used. Second, early, transient mitochondrial
morphological changes occur but are reversible. Third, the protective effects of partial DRP1
deficiency against neuroinflammation occur through a mechanism independent of mitochondrial
impairment, instead acting through decreased NF-κB activation and target gene regulation.
Lcn2 deletion reduces proinflammatory cytokine release from microglia
Lipocalin-2 is increasingly recognized as a potent inducer of neuroinflammation12, 14, 40. Our data
showing DRP1 deficiency reduces both NF-κB activity and lipocalin-2 levels suggest that a
DRP1–NF-κB– lipocalin-2 axis contributes to the inflammatory response. To determine whether
Lcn2 expression correlates with the production of proinflammatory cytokines, primary microglia
from Lcn2+/+, Lcn2+/-, and Lcn2-/- mice were treated with LPS, and cytokine levels in the
conditioned medium were measured by multiplex ELISA. As shown in Fig. 5a, basal secretion of
lipocalin-2 in Lcn2+/+ microglia was 5.8-fold higher than in Lcn2+/- microglia (p = 0.011) and
undetectable in Lcn2-/- cells. Such genotypic differences were not observed in the basal levels of
IL-6, IL-1β, CXCL-10/IP10, MCP-1/CCL2, and IFN-γ (Fig. 5a), indicating that Lcn2 deletion
selectively affects Lcn2 itself rather than general cytokine production. Upon LPS treatment,
proinflammatory cytokine secretion increased markedly in Lcn2+/+ microglia. Lcn2 deletion
attenuated these responses in a gene-dose–dependent manner (Fig. 5a). Partial loss of Lcn2
(Lcn2+/-) significantly reduced lipocalin-2, IL-6, and IL-1β, whereas complete deletion (Lcn2−/−)
produced broader suppression of additional cytokines (Fig. 5a). These data support a critical role
for lipocalin-2 in amplifying microglial inflammatory responses.
Given that DRP1 deficiency reduces neuroinflammation by decreasing NF-κB recruitment to
its target genes, we next asked whether the reduced inflammatory responses in Lcn2-KO
microglia might also reflect diminished NF-κB promoter recruitment. To address this question, we
performed ChIP assays to measure NF-κB p65 binding to the Lcn2 promoter using Lcn2+/+, Lcn2+/-
, and Lcn2-/- microglia. Surprisingly, NF-κB p65 enrichment at the Lcn2 promoter was inversely
correlated with Lcn2 expression levels (Fig. 5b), being highest in Lcn2−/− cells. We hypothesized
that this increased binding represents a compensatory attempt by NF-κB to upregulate Lcn2
expression. However, because exons 2–5 of the Lcn2 gene are deleted in the Lcn2−/− mice, NF-
κB binding cannot drive Lcn2 transcription; instead, NF-κB becomes sequestered, or “hijacked,”
at the nonfunctional Lcn2 promoter. If this model is correct, sequestration of NF-κB at the Lcn2
promoter should reduce its availability to bind other proinflammatory genes. To test this
hypothesis, we examined NF-κB p65 recruitment to the promoters of Cxcl10 and Il6, both
canonical NF-κB targets (Fig. 4a). Consistent with our hypothesis, NF-κB binding to these
promoters was markedly reduced in Lcn2+/− and Lcn2−/− microglia compared with Lcn2+/+ microglia
(Fig. 5b). These results indicate that the blunted inflammatory response observed in Lcn2-KO
microglia is due, at least in part, to sequestration of NF-κB at the inactive Lcn2 promoter, thereby
limiting its recruitment to other proinflammatory genes. Together, these results identify a
previously unrecognized mechanism by which loss of Lcn2 reduces neuroinflammation.
LPS induces upregulation and nuclear translocation of DRP1
Based on our observations that DRP1-KO reduced the expression of proinflammatory genes
(Figs. 1-2), we hypothesized that the reduced binding of NF-ĸB p65 to the promoter of Lcn2 was
due to lower expression of Rela, the gene encoding NF-ĸB p65. Indeed, qPCR analysis showed
significantly reduced Rela expression in Dnm1l+/- mice compared with WT littermates (Fig. 2a). It
has been reported that nuclear DRP1 accumulation occurs in lung adenocarcinoma might
contribute to hypoxia-induced drug resistance41. Although DRP1 is classically described as a
cytosolic and mitochondrial protein25, these observations raise the possibility that DRP1 may
translocate to the nucleus under certain conditions. Together, such prior research led us to
hypothesize that DRP1 might directly regulate transcription of proinflammatory genes.
To test this hypothesis, we used immunofluorescence and Imaris 3D reconstruction to quantify
nuclear (DRP1ᴺᵘᶜ, red) versus cytoplasmic (DRP1ᶜʸᵗᵒ, yellow) DRP1 in primary microglia and in
microglia from LPS-treated mice (Fig. 6a–d). Even in vehicle-treated controls, a low level of
nuclear DRP1 was detected, an unexpected observation that, to our knowledge, has not been
previously reported and it raises the question of why DRP1 is in the nucleus. Upon LPS exposure,
nuclear DRP1 increased 3.8-fold in primary microglia (p = 0.0084), while cytoplasmic DRP1
increased 2.3-fold (p = 0.0001) (Fig. 6a–b). Consistent results were observed in vivo: microglia in
the substantia nigra of LPS-treated mice exhibited a 3-fold increase in DRP1ᴺᵘᶜ (p = 0.0009) and
a 1.5-fold increase in DRP1ᶜʸᵗᵒ (p = 0.0479) relative to vehicle-treated controls (Fig. 6c–d). These
results indicate that LPS stimulation promotes both nuclear translocation of DRP1 and an overall
increase in DRP1 protein levels.
To quantitatively validate these observations, we performed capillary immunoblotting on
nuclear and cytosolic fractions of Dnm1l+/+ and Dnm1l+/- microglia with or without LPS treatment.
Fraction purity was confirmed by probing nuclear fractions for GAPDH (cytosolic marker) and
cytosolic fractions for Histone H3 (nuclear marker), with no evidence of cross-contamination
(Supplementary Fig. 5). In agreement with the imaging results, LPS increased DRP1ᴺᵘᶜ and
DRP1ᶜʸᵗᵒ levels by 2.5-fold (p = 0.0058) and 1.7-fold (p = 0.0072), respectively, in Dnm1l+/+
microglia (Fig. 6e–f). These increases were absent in Dnm1l+/- microglia, demonstrating that
partial DRP1 deficiency prevents LPS-induced DRP1 upregulation and nuclear enrichment.
Similar observations were made using whole cell extracts (Supplementary Fig. 6). Collectively,
these data show that LPS induces both upregulation and nuclear translocation of DRP1 in
microglia, and that this process is impaired in DRP1-deficient cells. These results support a model
in which nuclear DRP1 contributes to the transcriptional activation of proinflammatory genes.
DRP1 is recruited to the promoter region of Rela gene upon LPS exposure
Given that Rela levels were lower in Dnm1l+/- mice compared with their Dnm1l+/+ littermates
following LPS treatment (Fig. 2a), we hypothesized that Rela might be transcriptionally regulated
by DRP1. To test this hypothesis, we performed ChIP assays in WT microglia to determine
whether DRP1 is recruited to the Rela promoter after LPS exposure. Because DRP1 was not a
canonical transcription factor whose binding consensus motif was previously identified, we
designed seven primer pairs (P1–P7) to span ~2000 bp of the Rela promoter (Fig. 7a;
Supplementary Data 2). Strikingly, DRP1 enrichment was significantly increased at a 255 bp
region amplified by primer set P7 in LPS-treated microglia compared with vehicle controls (p <
0.0001; Fig. 7b). No enrichment was detected in IgG controls, and no DRP1 recruitment was
observed at regions amplified by P1–P6 (Fig. 7b). These results indicate that LPS exposure
induces selective recruitment of DRP1 to a promoter region proximal to the Rela transcription
start site.
To further validate the transcriptional regulation of Rela by DRP1, we cloned the 255 bp
DRP1-binding region identified by ChIP into the pGL4.20[luc2/Puro] luciferase reporter vector
(Fig. 7c). This construct, along with a promoterless control vector, was transfected into mouse
neuronal cells in the presence of either Dnm1l-specific siRNA or a scrambled control.
Untransfected cells were included as an additional control. As shown in Fig. 7d, luciferase activity
in cells driven by the DRP1-binding region was markedly increased (p < 0.0001), whereas those
with the promoterless vector or no transfection showed no activity. Silencing Dnm1l significantly
reduced luciferase activity compared with the scrambled control (p = 0.0048), consistent with
DRP1-dependent transcriptional activation. Dnm1l knockdown efficiency was 71.21 ± 0.68%
(Supplementary Fig. 7). These results demonstrate that DRP1 directly regulates the Rela
promoter in a transcriptionally active manner.
Finally, to determine the temporal relationship between DRP1 and NF-κB p65 nuclear
accumulation, we performed a time-course analysis following LPS stimulation. Nuclear DRP1
increased as early as 0.5 h after LPS exposure, whereas nuclear NF-κB p65 began to increase
at 3 h (Fig. 7g–i), indicating that DRP1 translocates to the nucleus prior to NF-κB p65. We did not
observe a significant increase in cytosolic NF-κB p65 levels up to 6 h after LPS treatment,
supporting the notion that DRP1 promotes NF-κB p65 nuclear translocation rather than retention
in the cytosol. Fraction purity was confirmed by probing nuclear fractions for GAPDH and cytosolic
fractions for Histone H3 (Supplementary Fig. 8).

Methods
Animal models and LPS treatment
All mice used in this study were bred, maintained, and characterized in the animal care facility
at Florida International University (FIU). Mice were housed under a standard 12 h light/12 h dark
cycle at controlled ambient temperature and humidity. All procedures were approved by, and
conducted in accordance with, the FIU Institutional Animal Care and Use Committee (Protocol
Approval #: IACUC-24-088-CR01).
Dnm1l-deficient mouse models. The generation of Dnm1l+/- mice with germline deletion of exon
2 of the Dnm1l gene has been described in our recent publication24. To generate conditional DRP1
knockout mouse lines, we contracted the European Conditional Mouse Mutagenesis Consortium
(EUCOMM) to produce Dnm1l-Loxp+/+ mice using “knockout-first” technology65. In this approach,
the tm1a allele, containing an IRES:lacZ gene-trap cassette and a floxed promoter-driven neo
cassette inserted into an intron of Dnm1l, was crossed to a FLP recombinase–expressing mouse
to convert the tm1a allele into the conditional tm1c24. The resulting Dnm1l-Loxp+/+ (tm1c) mice
were crossed with either Cx3Cr1-CreERT2+/-mice (#021160, Jackson Laboratory) or
Aldh1L1Cre/ERT2+/- mice (#031008, Jackson Laboratory) to generate Cx3Cr1-CreERT2+/-:Dnm1l-
Loxp+/- mice (“Microglia-KO”) and Aldh1L1Cre/ERT2+/-:Dnm1l-Loxp+/- (“Astrocyte-KO”) mice,
respectively. Dnm1l-Loxp+/- littermates were used as controls. Cre recombination was induced by
intraperitoneal injection of tamoxifen (75 mg/kg, dissolved in corn oil; Sigma T2859) for five
consecutive days. LPS treatment was performed three weeks after tamoxifen injection to ensure
efficient Dnm1l knockout.
α-Synuclein transgenic and DRP1-deficient double mutant mice. C57BL/6N-Tg(Thy1-
SNCA)15Mjff/J mice (#017682, Jackson Laboratory, Dnml1+/+:SNCA+/-) were crossed with
Dnm1l+/-:SNCA-/- (heterozygous DRP1-KO) mice to generate Dnm1l+/-:SNCN+/- double-
heterozygous mice. Dnml1+/+:SNCA-/- littermates served as controls.
Lcn2-knockout mice. B6.129P2-Lcn2tm1Aade/AkiJ (Lcn2-/-) mice (#024630, Jackson
Laboratory) were crossed with C57BL/6J mice to generate Lcn2+/- mice. Lcn2+/- mice were paired
with Lcn2+/- mice to maintain the colony.
LPS treatment. Three-month-old mice received a single intraperitoneal (i.p.) injection of LPS
(5 mg/kg; E. coli O111:B4, Sigma L4391) or saline (control). Male and female mice were
randomized into treatment groups. Mice were euthanized 6 hours after injection. This regimen
was selected because it induces rapid and sustained microglial activation in the substantia nigra
without causing dopaminergic neurodegeneration at this early time point32, 33. Brains were micro-
dissected by regions, snap-frozen or fixed for subsequent analysis.
NanoString codeset
The reporter codeset used was the nCounter Mouse Neuroinflammation Panel (cat. no.
115000237, NanoString Technologies), which measures 757 target genes spanning core
neuroinflammatory pathways and processes, along with 13 internal reference genes for data
normalization.
RNA extraction and NanoString nCounter analysis
Total RNA was extracted from mouse ventral midbrain using TRIzol (Thermo Fisher Scientific)
according to the manufacturer’s instructions. Seventy-five nanograms of total RNA per sample
was used for mRNA expression analysis on the NanoString nCounter SPRINT Profiler
(NanoString Technologies). Target mRNAs were hybridized to reporter-capture probe pairs at 65
°C for 20 h, following the manufacturer's protocol. The hybridized probe–target complexes were
subsequently aligned and immobilized in the nCounter cartridge, which was loaded into the
nCounter system for imaging and data collection. Gene expression levels were exported in
comma-separated value format. Raw digital counts were analyzed using nSolver v4.0 software
(NanoString Technologies). Differential gene expression analysis was performed using a
generalized linear model appropriate for count-based data. Statistical significance was
determined using model-based p-values, with multiple-testing correction performed using the
Benjamini–Hochberg method.
Quantitative RT-PCR
For reverse transcription, 1 μg of total RNA was converted to complementary DNA (cDNA) using
the iScript Reverse Transcription Supermix (Bio-Rad). qPCR was performed using TaqMan Fast
Advanced Master Mix (Thermo Fisher Scientific) and gene-specific TaqMan assays on a
QuantStudio 6 real-time PCR system (Thermo Fisher Scientific). The following mouse Taqman
assays were used: Lcn2 (ID: Mm01324470_m1), Nlrp3 (ID: Mm00840904_m1), Il6 (ID:
Mm00446190_m1), Il1b (ID: Mm00434228_m1) , Tnfα (ID: Mm00443258_m1), Nos2 (ID:
Mm00440502_m1), Rela (ID: Mm00501346I_m1), and Gapdh (ID: Mm99999915_g1). Gapdh
served as the internal control. Thermal cycling conditions were: 50 °C for 2 min, 95 °C for 2 min
and 40 cycles of 95 °C for 1 s and 60 °C for 20 s. Relative quantification was calculated using the
2-ΔΔCT method.
Meso Scale Discovery (MSD) multiplex immunoassay
Dmn1l+/- and Dmn1l+/+ mice (3-months old) were treated with LPS (5 mg/kg, i.p.) or saline control
for 6h before ventral midbrains were collected. Tissues were weighed and homogenized in 1x
Tris lysis buffer (150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 20 mM Tris-HCl, pH
7.5) supplemented with protease inhibitors at a ratio of 75 mg tissue per 0.5 mL ice-cold buffer.
Samples were briefly sonicated, incubated on ice for 30 min, and centrifuged at 20,000 x g for 10
min at 4°C. Supernatants were collected, and protein concentrations were determined using the
BCA assay. For in vitro studies, primary microglia isolated from newborn pups were plated in 96-
well plates and treated with 100 ng/mL LPS or vehicle for 6 h. After treatment, conditioned media
containing secreted cytokines were collected and transferred to new plates. Pro-inflammatory
cytokine levels in tissue lysates and conditioned media were measured using a custom U-PLEX
mouse panel (lipocalin-2, IL-6, IL-1β, TNF-α, IP-10, MCP-1/CCL2, and IFN-γ) on the Meso Scale
Discovery (MSD) platform following the manufacturer’s instructions. Plates were read using the
MSD QuickPlex SQ 120, and data were analyzed with Discovery Workbench software.
DAB staining and Sholl analysis
Mice were perfused and fixed as previously reported66. Mice were perfused with 0.9% saline
followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer. Brains were removed and
post-fixed in 4% PFA overnight at 4 °C. Tissues were cryoprotected by sequential overnight
incubation in 15% and 30% sucrose in 0.1 M phosphate buffer, then snap-frozen in 2-
methylbutane at –55 °C. Frozen brains were embedded in Optimal Cutting Temperature (OCT)
and cryosectioned at 30 µm using a CryoStar NX70 cryostat (Thermo Fisher Scientific). Coronal
sections were collected free-floating in 0.1 M Tris-buffered saline (TBS).
Midbrain sections were washed in TBS and incubated in 10% methanol + 0.3% H₂O₂ for 10
min at room temperature to quench endogenous peroxidases. Sections were then blocked in 5%
normal goat serum (NAGS) and 0.1% Triton X-100 in TBS for 1 h at room temperature, followed
by overnight incubation at 4 °C with primary antibody against IBA1 (1:1000, cat. no. 019-19741,
FUJIFILM Wako Chemicals) prepared in 2% NGS and 0.1% Triton X-100 in TBS.
After washing with 0.1M TBS, sections were incubated for 1 h at room temperature with
biotinylated goat anti-rabbit IgG (1:200, cat. no. BA-1100, Vector Laboratories) in 2% NGS/0.1M
TBS. Sections were then washed and incubated with ABC reagent (Vectastain ABC kit, cat. no.
PK-4000, Vector Laboratories) for 1 h, followed by development with 3,3′-diaminobenzidine (DAB)
substrate (cat. no. SK-4100, Vector Laboratories) for 10 min.
Slides were rinsed in water and dehydrated through graded ethanol (70%, 90%, 100%).
Coverslips were applied using DPX mounting medium (Sigma). Brightfield images were obtained
using a Nikon microscope with a 100× objective. Sholl analysis of microglial morphology was
performed using the Sholl Analysis plug-in for Fiji (http://fiji.sc/Sholl_Analysis) as previously
described67. Thirty microglia located in the substantia nigra were analyzed per group.
Immunofluorescence-laser microdissection (IF-LMD)
Serial coronal sections of midbrains containing the substantia nigra (SN, 10 µm) were cut on a
CryoStar NX70 cryostat (Thermo Fisher Scientific). Eight sections per brain hemisphere were
mounted onto PEN membrane slides (4 µm; cat. no. 11600288, Leica) for IF-LMD, as previously
described24. Sections were fixed in cold acetone for 5 min, rinsed in DEPC-treated 1× PBS, and
blocked with 5% normal goat serum (NGS) for 30 min at room temperature. Sections were then
incubated with primary antibodies for 1 h, followed by secondary antibodies for 30 min at room
temperature, with DEPC-treated PBS washes between each step. After counterstaining with DAPI
(10 µg/mL) for 5 min, sections were dehydrated through graded alcohols (30 s each) and air-
dried. Primary antibodies used were: Chicken anti-tyrosine hydroxylase (1:200, Abcam 76442),
Rabbit anti-IBA1 (1:100, Wako 019-19741), Mouse anti-GAD67 (1:50, Millipore MAB5406), and
Chicken anti-GFAP (1:200, Thermo Fisher PA1-10004). Secondary antibodies (all 1:100 dilution)
included: Alexa Fluor 568 goat anti-chicken (Thermo Fisher A11041), Alexa Fluor 488 goat anti-
rabbit (Thermo Fisher A11008), and Alexa Fluor 488 goat anti-mouse (Thermo Fisher A11029).
Immunostained dopaminergic neurons (SNpc), GABAergic neurons (SNpr), and microglia and
astrocytes (substantia nigra) were isolated by laser microdissection using the Leica LMD6 system.
A total of 40 cells per cell type were collected for downstream analyses.
Reverse transcription and cDNA preamplification using Smart-seq2 method
Laser microdissected cells (40 cells per cell type) were collected into 0.2-mL thin-wall PCR tubes
containing 2 μL of mild hypotonic lysis buffer (0.2% Triton X-100 and 2 U/μL RNase inhibitor;
Clontech). Cell lysates were processed for reverse transcription and cDNA preamplification
following the Smart-seq2 protocol68, 69. For first-strand synthesis, lysates were mixed with 1 μL
oligo-dT primer (10 μM; Integrated DNA Technologies) and 1 μL dNTP mix (10 mM; Thermo
Fisher Scientific), denatured at 72 °C for 3 min, and immediately cooled on ice. Reverse
transcription was performed using SuperScript II reverse transcriptase (Thermo Fisher Scientific)
with the following program: 42 °C for 90 min, 10 cycles of 50 °C for 2 min and 42 °C for 2 min,
and 70 °C for 15 min for enzyme inactivation. Subsequently, cDNA preamplification was carried
out using KAPA HiFi HotStart ReadyMix (Roche) under the following conditions: 98 °C for 3 min,
24 cycles of 98 °C for 15 s, 67 °C for 20 s, and 72 °C for 6 min, followed by a final extension at
72 °C for 5 min. PCR products were purified using AMPure SXP beads (Beckman Coulter)
according to the manufacturer’s instructions and eluted in 15 μL TE buffer. The resulting
preamplified cDNA was used for downstream qPCR analysis as described above.
Primary mouse microglia
Primary microglia were isolated from postnatal day 0–1 mouse pups as previously described70.
Briefly, following decapitation, whole brains were removed, meninges were carefully dissected
away, and tissues were incubated in 0.25% trypsin at 37 °C for 15 min with gentle inversion every
5 min. The digested tissue was triturated and passed through a 70-µm cell strainer before plating
into culture flasks. Mixed glial cultures were maintained for 16 days in DMEM/F12 supplemented
with 10% heat-inactivated fetal bovine serum (HI-FBS), 2 mM L-glutamine, 1 mM sodium
pyruvate, 1% non-essential amino acids, and 1% penicillin–streptomycin. A full medium change
was performed on day 6 after initial plating. On day 16, microglia were isolated from the mixed
glial cultures using a CD11b immunomagnetic positive selection kit (cat. no. 18970, STEMCELL
Technologies) and plated for experiments. For all treatments, HI-FBS was reduced to 2%.
Microglial purity (>95%) was confirmed by Iba1 immunofluorescence.
Adult microglia isolation and mitochondrial respiration assay
Wild-type mice (2–4 months old) were injected with LPS (5 mg/kg, i.p.) or saline. Six hours after
injection, microglia were isolated using a modified gradient centrifugation protocol71, 72. Mice were
perfused with ice-cold sterile 0.9% saline, and brains were rapidly removed following decapitation
and placed into ice-cold Hibernate A medium supplemented with B27, GlutaMAX, penicillin–
streptomycin, and DNase I (HABG)72. Brains were transferred onto sterile filter paper pre-wetted
with HABG on a cooled platform and finely minced with a sterile scalpel. Minced tissue was then
placed into a dissociation solution (DMEM/F12 containing 1 mg/mL papain, 1.2 U/mL dispase II,
and 20 U/mL DNase I) and incubated at 30 °C for 20 min, with gentle inversion every 5 min. After
digestion, a neutralization solution was added and the tissue suspension was transferred to new
tubes containing 2 mL HABG.
Tissues were gently triturated with a fire-polished 9-inch Pasteur pipette, and the resulting cell
suspension was collected and filtered through a 70-µm strainer. The filtrate was centrifuged at
400 x g for 5 min at 4 °C, and the pellet was resuspended in 6 mL HABG. The cell suspension
was carefully layered onto a freshly prepared four-layer OptiPrep density gradient (7.4%, 9.9%,
12.4%, and 17.4% OptiPrep in Hibernate A/B27)71.
Gradients were centrifuged at 800×g for 30 min at 22 °C, using minimal
acceleration/deceleration (setting 0). The upper layers were aspirated, and the microglia-enriched
lower fractions71 were collected and diluted in 5 mL cold HABG, followed by centrifugation at
400×g for 10 min to remove residual OptiPrep. Pellets were resuspended, counted, and plated
into Seahorse assay plates. Plates were centrifuged at 1,000×g for 15 min to promote adhesion,
then topped with assay medium and equilibrated in a non-CO₂ incubator for 30 min. Mitochondrial
respiration was assessed using a standard Mito Stress Test on the Agilent XFe96 Extracellular
Flux Analyzer, with extended basal measurements.
Mitochondrial respiration assay
Mitochondrial respiration was assessed using Agilent XFe 96 Extracellular Flux Analyzer (Agilent
Inc) as previously reported70. Briefly, primary microglia isolated from Dnm1l+/+ and Dnm1l+/- mouse
pups were seeded into XF Cell Culture Microplates and treated the following day with LPS (100
ng/mL) for 2 h, 6 h, or 24 h. On the day of the assay, cells were washed twice with XF assay
medium (DMEM supplemented with 1 mM pyruvate, 2 mM GlutaMAX, 2 mM glucose, and 2 mM
HEPES, pH 7.2). Plates were then equilibrated in a non-CO₂ incubator for 30 min before being
loaded into the Analyzer. A standard XF Cell Mito Stress Test was performed, with sequential
injections of oligomycin, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), and
rotenone/antimycin A to evaluate ATP-linked respiration, maximal respiration, and non-
mitochondrial oxygen consumption, respectively.
Immunostaining
Primary microglia. Primary mouse microglia were cultured on poly-D-lysine–coated
borosilicate coverslips in 24-well plates. After treatment with LPS (100 ng/mL) for 2, 6, or 24 h,
cells were fixed and immunostained as previously described70. Primary antibodies included:
Rabbit anti-TOM20 (1:1000, Abcam ab186735), Mouse anti-DRP1 (1:500, BD Biosciences
611113), Rabbit anti-DRP1 (1:500, Novus NB110-55288), and Goat anti-IBA1 (1:500, Novus
NB100-1028). Corresponding Alexa Fluor 488, 594, or 633 secondary antibodies (Invitrogen)
were used at 1:1000. Coverslips were incubated with primary antibodies overnight at 4 °C on an
orbital shaker, followed by secondary antibody incubation at room temperature for 1 h. Mounted
coverslips were prepared using ProLong™ Gold Antifade Mountant with DAPI (Thermo Fisher
Scientific).
Brain tissue sections. Mice were perfused with 0.9% saline followed by 4% paraformaldehyde
(PFA) in 0.1 M phosphate buffer 6 h after LPS exposure. Brains were post-fixed, snap-frozen in
2-methylbutane, embedded in OCT, and cryosectioned at 30 µm. Free-floating coronal sections
were collected in 0.1 M TBS. Sections were blocked using the Mouse-on-Mouse (M.O.M.) reagent
(Vector Laboratories, BMK2202) and incubated with the following primary antibodies: Mouse anti-
DRP1 (1:500, BD Biosciences 611113), and Rabbit anti-IBA1 (1:1000, FUJIFILM Wako 019-
19741). Alexa Fluor secondary antibodies (Invitrogen) were used at 1:1000. After
immunostaining, sections were incubated with DAPI (5 µg/mL) for 10 min, washed, and mounted
with ProLong™ Gold Antifade Mountant. All fluorescent images were captured using an Olympus
Fluoview FV10i confocal microscope equipped with a 60× objective.
Mitochondrial morphology and network analysis
Mitochondrial morphology and network structure were quantified using the Mitochondrial Network
Analysis (MiNA) plugin in Fiji, as previously described24. Mitochondria were visualized by TOM20
immunofluorescence (Abcam ab186735) and imaged using an Olympus Fluoview confocal
microscope. Z-stack images were used for all MiNA analyses. MiNA-derived parameters included:
Mean rod/branch length: the average length of all mitochondrial rods and branches. Mean
branches per network: the average number of branches within each mitochondrial network.
Individual mitochondrial morphology was further assessed in Fiji as previously reported70. Two
metrics were quantified: Roundness = 4 × (Area) / (π × [Major axis]²) and Aspect ratio = Major
axis / Minor axis. Values for both parameters approach 1 as mitochondria become more circular.
Quantification using Imaris
DRP1 nuclear translocation in primary microglia and in microglia within the substantia nigra of
adult mouse brains was quantified using Imaris (Oxford Instruments). Z-stack confocal images
were acquired using a 60× objective lens with 3× optical zoom. Briefly, nuclear and cellular 3D
surfaces were reconstructed from the blue (nuclei) and cyan (cell surface) channels using the
“Surfaces” module. DRP1 puncta were detected from the red fluorescence channel using the
“Spots” module, with identical threshold settings applied across all treatment groups. DRP1Nuc
was defined as the number of DRP1 puncta located within the nuclear volume. DRP1Cyto was
defined as the number of DRP1 puncta located within the cytoplasmic volume, calculated as total
cellular puncta minus of DRP1Nuc.
Nuclear and cytoplasmic fractionation
To quantitatively assess DRP1 nuclear enrichment following LPS exposure, nuclear and
cytoplasmic extracts were prepared from LPS-treated primary mouse microglia; vehicle-treated
cells served as controls. Cells were harvested into 100 μL Cytoplasmic Extract Buffer (10 mM
HEPES, pH 7.6; 60 mM KCl; 1 mM EDTA; 0.075% NP-40; 1 mM DTT; 1 mM PMSF) and incubated
on ice for 10 min to permeabilize the plasma membrane. Lysates were centrifuged at 200 × g for
4 min at 4 °C to separate the cytoplasmic fraction (supernatant) from the intact nuclei (pellet). The
supernatant was collected as crude cytoplasmic lysate. Nuclear pellets were washed once with
Cytoplasmic Extract Buffer lacking NP-40, followed by centrifugation at 200 × g for 4 min at 4 °C.
Pellets were then resuspended in Nuclear Extract Buffer (20 mM Tris-HCl, pH 8.0; 420 mM NaCl;
1.5 mM MgCl₂; 0.2 mM EDTA; 0.5 mM DTT; 25% glycerol; 1 mM PMSF). Nuclear suspensions
were homogenized on ice using a Qsonica sonicator for 3 cycles of 30 s ON/30 s OFF at 60%
amplitude. Both crude cytoplasmic and nuclear suspensions were centrifuged at 21,000 × g for
30 min at 4 °C to remove debris. The resulting supernatants, cytoplasmic extracts and nuclear
extracts, were aliquoted and stored at –80 °C for subsequent analyses.
Capillary gel-free immunoblotting
Capillary immunoblotting was performed using the Jess™ system (ProteinSimple) with the 12–
230 kDa Fluorescence Separation Module (cat. no. SM-FL004), as previously described24. Protein
concentrations were quantified using the Micro BCA kit (cat. no. 23235, Thermo Fisher Scientific).
Cytoplasmic extracts, nuclear extracts, or whole-cell lysates (prepared in RIPA buffer) were
diluted in 0.1× sample buffer and mixed with 5× Fluorescent Master Mix (cat. no. PS-ST01EZ,
ProteinSimple) to final concentrations of 0.5 µg/µL and 1.2 µg/µL, respectively. Samples were
boiled at 95 °C for 5 min, and 3 µL of each sample were loaded into the capillary system.
Primary antibodies included: Rabbit anti-DRP1 (1:100, Novus NB110-55288), Rabbit anti–NF-κB
p65 (1:25, CST 8242), Rabbit anti–Histone H3 (1:100, Abcam ab1791), and Rabbit anti-GAPDH
(1:100, CST 2118). Anti-rabbit secondary antibodies were supplied through the appropriate
Detection Module (cat. no. DM001, ProteinSimple). The Protein Normalization Module (cat. no.
DM-PN02, ProteinSimple) was used for quantitative signal normalization. Data acquisition and
analysis were carried out using Compass software v6.0.
SDS-PAGE Immunoblotting
Mouse ventral midbrains were resuspended in RIPA lysis buffer and sonicated on ice using a
Qsonica sonicator for 5 cycles of 10 s ON/10 s OFF at 50% amplitude. Lysates were rotated at 4
°C for 30 min, followed by centrifugation at 13,000 × g for 15 min at 4 °C. Primary microglia were
lysed directly in RIPA buffer for 5 min on ice before centrifugation. Protein concentrations for all
samples were determined using the Pierce™ BCA Protein Assay (Thermo Fisher Scientific).
Proteins were resolved by 12% SDS-PAGE or 15% SDS-PAGE (for p62) and transferred onto
0.45-µm PVDF membranes (Immobilon-P, MilliporeSigma). Total protein levels were assessed
using Revert™ 700 Total Protein Stain (LI-COR Biosciences) according to the manufacturer’s
instructions. Membranes were blocked for 1 h at room temperature in Intercept® Blocking Buffer
(LI-COR Biosciences) and incubated overnight at 4 °C with the following primary antibodies:
Rabbit anti–pDRP1-Ser616 (1:1000, CST 4494), Rabbit anti–pDRP1-Ser637 (1:1000, GeneTex
GTX01567), Rabbit anti–NF-κB p65 (1:1000, CST 8242), Mouse anti–IκBα (1:1000, Novus
NB100-56507), and Rabbit anti-p62 (1:1000, MBL PM045). After washing with TBS-T,
membranes were incubated for 1 h at room temperature with the appropriate secondary
antibodies: Anti-rabbit IgG (H+L) DyLight® 800 (1:10,000; or 1:5000 for p62; CST 5151), Anti-
mouse IgG (H+L) DyLight® 800 (1:10,000; CST 5257). Blots were imaged using the LI-COR
Odyssey Imaging System, and band intensities were quantified using Image Studio™ Software
v6.0 (LI-COR Biosciences).
Chromatin immunoprecipitation (ChIP) assay
A total of 6 × 10⁶ primary microglia were treated with LPS (100 ng/mL) for 6 h; cells treated with
medium alone served as controls. After treatment, cells were washed twice with PBS containing
protease inhibitors and processed for the ChIP assay. For crosslinking, cultures were incubated
in medium containing 1% (w/v) formaldehyde (Sigma-Aldrich) at 37 °C for 15 min (NF-κB p65) or
30 min (DRP1). Crosslinking was quenched with 125 mM glycine for 5 min with shaking. Cells
were collected by scraping, centrifuged, washed twice with cold PBS plus protease inhibitors
(Pierce, Thermo Fisher Scientific), and resuspended in cell lysis buffer containing 1% SDS, 10
mM EDTA, 50 mM Tris-HCl (pH 8.0) and protease inhibitors. Lysates were incubated on ice for
10 min. Chromatin shearing was performed using a Qsonica sonicator at 4 °C for 30 cycles of 10
s ON/30 s OFF at 60% amplitude, resulting in cell lysis and DNA fragmentation. Lysates were
centrifuged at 18,000 × g for 10 min at 4 °C, and the clarified supernatant was collected. The
supernatant was diluted 10-fold with ice-cold ChIP dilution buffer (1% Triton X-100, 1.2 mM EDTA,
167 mM NaCl, 16.7 mM Tris-HCl, pH 8.0, with protease inhibitors). A 100-µL aliquot was set aside
as the Input.
The remaining supernatant was pre-cleared by incubation with sheared salmon sperm DNA–
coated protein A agarose beads (Pierce, Thermo Fisher Scientific) for 2 h at 4 °C with rotation.
The pre-cleared chromatin was then divided into equal aliquots and incubated overnight at 4 °C
with: IgG control (2 µg; CST #2729), Anti–NF-κB p65 antibody (2 µg; CST #8242), and Anti-DRP1
antibody (2 µg; CST #8570). Immune complexes were captured using sheared salmon sperm
DNA–coated protein A agarose beads for 2 h at 4 °C with rotation. Bead-bound complexes were
pelleted at 500 × g for 2 min and washed sequentially: twice with low-salt wash buffer (150 mM
NaCl), once with high-salt wash buffer (500 mM NaCl), and once with TE buffer. Chromatin
was eluted from the beads using freshly prepared elution buffer (1% SDS, 0.1 M NaHCO₃).
Crosslinks were reversed by incubation with 0.2 M NaCl at 65 °C overnight. Samples were then
treated with proteinase K at 45 °C for 2 h, followed by phenol–chloroform extraction and ethanol
precipitation. Purified DNA was resuspended in TE buffer and used for quantitative PCR analysis.
Quantitative real-time PCR for ChIP assay
Quantitative real-time PCR was performed using KAPA SYBR FAST qPCR Master Mix (Sigma)
in a 10-µL reaction containing purified ChIP DNA (NF-κB p65–bound, DRP1-bound, or IgG
control) and gene-specific primers (Supplementary Data 2). Reactions were run on a QuantStudio
6 real-time PCR system (Thermo Fisher Scientific) using the following cycling program: 95C for
3 min (1 cycle), 40 cycles of 95C for 3 s, Primer-specific-annealing temperature Ta
(Supplementary Data 2) for 20 s, 72C for 1 s. Ct values were obtained using QuantStudio
Software v1.3. Data analysis:
Fold enrichment was calculated by normalizing ChIP Ct values to the Input DNA. The normalized
ΔCt for each ChIP reaction was computed as: Δ𝐶𝑡 = 𝐶𝑡 −(𝐶𝑡 −
normalized ChIP ChIP Input
log⁡(Input dilution factor)). The Input dilution factor is defined as volume used for each IP/the
volume of Input saved for further analysis. Volume saved = 100 µL and volume used for each IP
= 900 µL. Thus, the Input dilution factor was 9 and the equation was Δ𝐶𝑡 = 𝐶𝑡 −
normalized ChIP ChIP
(𝐶𝑡 −log⁡(9)). The percentage of Input was calculated as: Input % = 2−Δ𝐶𝑡normalized ChIP ×100.
Input 2
The Input % value reflects the enrichment of NF-κB p65 or DRP1 (relative to IgG control) at the
promoter region of their target gene(s). Higher Input % values indicate greater transcription factor
or DRP1 binding.
Construction of mouse Rela promoter-driven reporter construct
Genomic DNA isolated from Dnm1l+/+ (WT) mice was used as the template for PCR amplification
of the DRP1-binding region within the mouse Rela promoter. The primer sequences were as
follows: sense, 5’- ggtaccACC TGC GGA GCT TGT AG -3’, and antisense,5’- aagcttGCT AAA
GTA AAG CCA TTC G -3’. The sense primer contained a KpnI restriction site, and the antisense
primer contained a HindIII site. PCR was performed using Platinum II Taq Hot-Start DNA
Polymerase (Invitrogen, cat. no. 14000012) according to the manufacturer’s instructions.
Amplified fragments were first ligated into the PCR2.1 vector (Invitrogen, cat. no. K203001).
Following digestion with the corresponding restriction enzymes and gel purification (Qiagen, cat.
no. 28704), the inserts were subcloned into the pGL4.20[luc2/Puro] vector (Promega, cat. no.
E6751). The DNA sequence of the final construct was confirmed by whole-plasmid sequencing
(Eurofins Genomics).
Cell transfection and luciferase assay
HT22 mouse hippocampal cells were plated at a density of 1 × 10⁴ cells per well in 96-well plates.
After 24 h, cells were transfected with 0.1 μg of either the pGL4.20[luc2/Puro] control vector or
the pGL4.20[luc2/Puro]-Rela construct in the presence or absence of Dnm1l-specific siRNA (50
nM) or a scrambled siRNA control using jetPRIME (SATORIUS, cat. no. 10100001), following the
manufacturer’s protocol. The siRNA reagents included SMARTpool siGENOME mouse Dnm1l
siRNA (Dharmacon/Horizon, cat. no. M-054815-01), which is a mixture of four siRNA targeting a
single gene to enhance efficiency and specificity of Dnm1l-knockdown. The siGENOME Non-
Targeting siRNA Control Pool (cat. no. D-001206), the scramble control, has a minimum of four
siRNAs designed to target no known genes in human, mouse or rat genes. Firefly luciferase
activity was measured 48h post-transfection using the ONE-Glo™ EX Luciferase Assay System
(Promega, cat. no. E8110), following the manufacturer’s instructions. Knockdown efficiency of
Dnm1l was confirmed by qRT-PCR analysis of Dnm1l mRNA levels.
Statistics
All values are presented as mean ± SEM. Differences between group means were analyzed using
t-tests or one-, two-, or three-way ANOVA, with different mouse genotypes, treatment, and or time
as independent factors. When ANOVA indicated a significant main effect or interaction, Tukey’s
post hoc test was used for pairwise comparisons. All datasets were evaluated for normality and
homogeneity of variance. When these criteria were not met, nonparametric tests were applied,
specifically the Kruskal–Wallis ANOVA followed by Dunn’s post hoc test. In all analyses, the null
hypothesis (H ) or the alternative hypothesis (H ) was accepted with an -error (false-positive) 
0 1
5% and a -error (false-negative)  20%, respectively. F-values, degrees of freedom, and
associated p-values for all analyses are reported in Supplementary Data 3. Volcano plots were
generated using the Positron integrated development environment (version 2025.11.0, build 234;
Posit PBC) with Bioconductor version 3.22 and the EnhancedVolcano package version 1.27.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Drp1 Induces Neuroinflammation via Transcriptional Regulation of NFkB\nThis manuscript by Lai al. presents compelling evidence for a non-canonical role of Drp1 regulating neuroinflammation.\nUsing transgenic mouse models of Drp1, the authors first identify lipocalin-2 as a key proinflammatory target of Drp1 in glial\ncells of the ventral midbrain regions following LPS stimulation. They further demonstrate that Drp1 is upregulated and\nrapidly translocated locates to the nucleus under these conditions. Notably, authors uncover that Drp1 directly binds to Rel\npromoter, thereby driving lipocalin-2 mediated neuroinflammatory responses. The study employes multiple methodologies\nand models, and the experimental design appears rigorous and well-executed. This work provides novel insights into\npreviously unreported function of Drp-1, suggesting its dual role in mitochondrial quality control and dampening\nneuroinflammation. These results have important implications for development of Drp1 targeted disease modifying therapies\nfor Parkinson’s and other related neurodegenerative diseases.\nSome questions and clarifications that should be addressed.\n1. Authors used a 5 mg/kg LPS injection for 6 hours to evoke neuroinflammation in the brain. While the rationale for this dose\nis briefly discussed in context of disentangling Drp1’s dual function in the Discussion section, a clear justification should\nalso be provided in the in methods section.\n2. In fig 3b, authors show LPS treatment increases Lcn2 mRNA level in microglia, astrocytes and dopaminergic neurons.\nThe underlying mechanism driving this upregulation warrants further discussion. Specifically, is this effect mediated by\nneuronal Drp1upregulation and translocation?\n3. Drp1 is known to undergo multiple post-translational modifications. Does LPS-induced Drp1 translocation to nucleus\nrequire any post-translational modifications?\n4. A discussion of the study’s limitations and potential future directions would be valuable. This could include technical\nconstraints, unanswered mechanistic questions, or border implications for therapeutic strategies for chronic\nneurodegenerative diseases.", "reviewer_2": "In this paper, Lai et al. presented relevant evidence indicating that DRP1, a protein regulator of mitochondrial fission,\ncontrols neuroinflammatory response by activating NF-kB. Apparently, this mechanism is produced by the protein lipocalin-2\nin microglia and astrocytes. These interesting studies propose a novel action of DRP1 on neuroinflammation and link\nmitochondrial function with inflammatory response in the brain. However, several points require clarification before any\npublication process.\nMayor comments\n-The authors declare: “Emerging evidence indicates the central role of neuroinflammation in neurodegenerative diseases\nsuch as Alzheimer’s disease (AD), Parkinson’s disease (PD)2”. However, this is not emerging evidence, which has been\nobserved and proposed for quite a while. The authors must clarify this information and reduce the enthusiasm of these\nstatements.\n-The authors must discuss the antioxidant factor NRF2's contribution to NF-KB regulation and its involvement in the\nmechanism explored in this manuscript. Several studies suggest that NRF-2 modulates the NF-κB response, decreasing\ninflammatory signaling. If we consider the scope of this paper, perhaps NRF2 could be playing a role, or at least this action\nshould be experimentally discarded.\n-Did the authors test Lcn2 levels in any AD mouse models? If not, it is better that the paper focus on PD effects related to\nLcn2 and not AD.\n-The authors need to include specific evidence regarding the neurodegenerative role of lipocalin 2 in PD.\n-As the authors have investigated DRP1 involvement in neuroinflammatory action, they should be testing the effects on\nmitochondrial dynamics. They need to show if the change in DRP1 affects the expression of the other mitochondrial dynamic\nregulators (Opa1, Mfn1/2, Fis1).\n-Also, the authors should specify how DRP1 activation induced by several phosphorylation statuses could be involved in the\nneuroinflammatory regulation induced by NF-kB activation. At least two specific phosphorylation residues (616 or 636) can\nbe affected by oxidative stress or other stressors important in neuroinflammation.\n-The analysis of mitochondrial dynamics presented in Fig.S3 must be augmented using the macro tool for Fiji-ImageJ MiNA\nanalysis.", "reviewer_3": "Lai and colleagues highlight a novel role for dynamin-related protein 1 (DRP1), previously known as a key mediator of\nmitochondrial fission and autophagy flux, now proposed to act as a transcription factor regulating the expression of the\nLipocalin 2 gene through the control of Rela (encoding NF-κB). The authors employ a comprehensive set of state-of-the-art\ntechniques, including both constitutive and inducible transgenic mouse models, digital PCR-based quantification of\nproinflammatory mRNA markers in ventral midbrain samples and laser-microdissected brain cells, high-resolution IMARIS\nconfocal imaging, and capillary immunoblotting employing samples from primary microglial cultures. Although the study\nsupports the identification of a novel function for DRP1 in neuroinflammation, there are several concerns that should be\naddressed prior to publication.\nGeneral Comments:\n1. Although the topic is briefly addressed in the Discussion, the manuscript would benefit from a more clearly defined\nconceptual framework focused on general neuroinflammation in which microglial cells are the driving force. Notably, acute\nintraperitoneal LPS administration does not elicit a brain-specific proinflammatory response and astrocytes findings are not\nexplored in depth. The extrapolation of the findings to other neurodegenerative conditions such as Alzheimer’s disease,\nParkinson’s disease, or aging appears premature and should be reconsidered. This is particularly relevant given that the\nstudy relies on a single, SNCA model of PD (Dnm1l+/-:SNCA+/- , that is not characterized in the study) and includes only\none experiment related to aging. The impact on dopaminergic neuronal survival in response to LPS is not addressed at all,\ntherefore the relevance in PD models is not analyzed. Limiting the scope of the discussion to the specific experimental\ncontext would strengthen the manuscript’s coherence and rigor.\n2. Please clarify the rationale for using partial instead of full knockout Dnm1l models. In this regard, a deeper\ncharacterization of the employed mutant mice with heterozygous Dnm1l knockout is needed. From the molecular\nperspective, it would be important to analyze Dnm1l expression levels, both at the mRNA and protein level in ventral\nmidbrain extracts, as well as the phosphorylation status of DRP1. Additionally, the expression of key components of the\nNFκB pathway (such as IKK activation, and the protein levels of IκBα, p50, and p65) should be assessed in comparison to\nwild-type controls. Related to mitochondrial physiology, it would be crucial to describe the impact of deleting one copy of\nDnm1l gene on mitochondrial activity in the brain.\nSpecific comments.\nFigure 1. To better illustrate the protective role of Dnm1l, please provide a more comprehensive description of all\ndysregulated transcripts comparing LPS-Dnm1l +/- vs LPS-Dnm1l +/+, not just the top ten or the table. Include those\nvalidated at the protein level (e.g., IL-6, IL-1β, TNFα) in next figures.\nFigure S3. To rule out mitochondrial misbalance is crucial to include positive control (e.g., rotenone or paraquat) to validate\nOCR respiration measures. Also, autophagy should be analyzed, especially considering previous work by the same group\nshowing that partial Dnm1l knockout improves autophagic flux independently of mitochondrial function.\nFigure 6. Regarding panel 6e, why are DRP1 nuclear levels similar between Dnm1l+/- and saline-treated controls? There is\na reduction only in the cytosolic fraction. This analysis should be repeated in whole-cell extracts to assess total protein\nlevels (this is closely related to the second general comment). Figure S4. An explanation addressing why Histone H3 levels\nare reduced in nuclear extracts from mutant mice compared to wild-type controls could be added.\nFigures 6 and 7. Since LPS can activate NFκB independently of DRP1, it is crucial to show that the identified DRP1 motif is\nfunctional. Therefore, the proposed DRP1 nuclear translocation and binding to Rela regulation sites could be strengthened\nby: Showing p65 protein levels in both cytosol and nucleus (Figure 6e–f), highlighting the sequence of the DRP1 motif\nidentified by P7 primers (Figure 7), demonstrating that DRP1 binding leads to increased Rela transcription, to support the\nclaim made in line 327.\nMinor Points:\nLine 38: “Low-grade chronic inflammation” better describes the inflammatory state in neurodegenerative diseases.\nLine 65: Please elaborate on the neurotoxic role of Lipocalin-2.\nLine 71: Use “levels are” instead of “levels is”.\nLine 119: Replace “upregulation” with “was upregulated”.\nLine 117: Clarify whether the global DRP1-KO mouse refers to a previous study (add reference) or unpublished data.\nLine 195: The sentence appears to be cut off (please revise).\nLine 432: The sentence is split across two lines (please correct).\nLPS (cat# L4391, Sigma-Aldrich): Include the strain type to improve reproducibility.\nSupplementary Table 1: The PDF formatting is unclear (please improve the table layout for readability maybe in an Excell\nfile if possible).\nFigure S1a: Add a scale bar to indicate magnification.", "reviewer_4": "Lai reports a novel mechanism underlying DRP1-mediated inflammation induced by LPS. Reduced DRP1 expression\nsignificantly suppresses LPS-induced inflammation, including the proinflammatory molecule lipocalin 2. Upon LPS\nstimulation, DRP1 translocates from the cytosol to the nucleus, where it binds to the promoter region of Rela (encoding NF-\nκB), activating its gene products and downstream inflammatory cytokines. These findings highlight the important role of\nproinflammatory lipocalin 2 in the brain.\nTogether, this study reveals a previously unrecognized function of DRP1 in mediating neuroinflammation via the NF-κB–\nlipocalin 2 axis. The experiments are well-designed and clearly presented. Only minor revisions are suggested.\nSuggestions:\n• Please expand the discussion on the significance and rationale for using the LPS-induced model, particularly in relation to\nDRP1-mediated inflammation. How well does this model reflect the in vivo mechanisms of neuroinflammation?\n• It would also strengthen the manuscript to discuss the outcomes of additional models (if available) that explore the\nrelationship between DRP1-mediated mitochondrial morphology changes, neuroinflammation, and neurodegeneration. This\ncould provide further support for the role of DRP1 in disease progression and therapeutic targeting."}, "author_response": "Response to Reviewers\nRe: Manuscript resubmission (NCOMMS-25-49663-T)\nWe would like to express our sincere gratitude to the reviewers for their constructive, insightful, and\nsupportive feedback, which has greatly helped us improve the manuscript. Our detailed point-by-point\nresponses to the reviewers’ comments are provided below. For clarity and convenience, new data\nhave been included in this response letter and referenced where they have been incorporated into the\nrevised manuscript. Text revisions in the manuscript are highlighted in red.\nReviewer #1\nThis manuscript by Lai al. presents compelling evidence for a non-canonical role of Drp1 regulating\nneuroinflammation. Using transgenic mouse models of Drp1, the authors first identify lipocalin-2 as a\nkey proinflammatory target of Drp1 in glial cells of the ventral midbrain regions following LPS\nstimulation. They further demonstrate that Drp1 is upregulated and rapidly translocated locates to the\nnucleus under these conditions. Notably, authors uncover that Drp1 directly binds to Rel promoter,\nthereby driving lipocalin-2 mediated neuroinflammatory responses. The study employes multiple\nmethodologies and models, and the experimental design appears rigorous and well-executed. This\nwork provides novel insights into previously unreported function of Drp-1, suggesting its dual role in\nmitochondrial quality control and dampening neuroinflammation. These results have important\nimplications for development of Drp1 targeted disease modifying therapies for Parkinson’s and other\nrelated neurodegenerative diseases.\nSome questions and clarifications that should be addressed.\n1. Authors used a 5 mg/kg LPS injection for 6 hours to evoke neuroinflammation in the brain. While\nthe rationale for this dose is briefly discussed in context of disentangling Drp1’s dual function in the\nDiscussion section, a clear justification should also be provided in the in methods section.\nResponse: The in vivo LPS regimen used in this study was selected partly based on previous\nstudies, which show rapid and sustained microglia activation in the substantia nigra. Moreover, at this\ntreatment time-point, LPS does not cause dopaminergic neurodegeneration. Prior studies using this\nregimen show that microglial activation emerges within hours of peripheral LPS injection, but\ndopaminergic neurodegeneration in the substantia nigra develops much later, approximately seven\nmonths to observe modest loss (∼23%) and ten months to detect more pronounced degeneration\n(39–47%). Thus, this model reflects an early, pre-degenerative stage of neuroinflammation. We\nbelieve this model represents an early stage of neuroinflammation preceding cell death in PD. This is\ncritical to detect early translocation of DRP1 to the nucleus to initiate neuroinflammation. Additionally,\nwe found that this paradigm markedly increased pro-inflammatory cytokines in the VMB, yet\nmitochondrial respiration in microglia remained intact. This justification and relevant references have\nbeen elaborated more in Discussion (page 16-17) and added to the Methods section (page 23) as\nsuggested.\n2. In fig 3b, authors show LPS treatment increases Lcn2 mRNA level in microglia, astrocytes and\ndopaminergic neurons. The underlying mechanism driving this upregulation warrants further\ndiscussion. Specifically, is this effect mediated by neuronal Drp1upregulation and translocation?\nResponse: Our results show that Lcn2 expression increased in microglia, astrocytes, and DA\nneurons, but not GABA neurons, following LPS exposure. The lack of induction in GABA neurons\nsuggests cell-type-specific differences in Lcn2 inducibility, which warrants future studies. Combined\nwith the demonstration that LPS induced DRP1 translocation in microglia, we believe that the\nupregulation of Lcn2 observed in cell type is unlikely to originate from neuronal sources alone, and the\nelevated Lcn2 detected in vivo likely arises predominantly from glial sources. This discussion has\nbeen added to page 23.\n3. Drp1 is known to undergo multiple post-translational modifications. Does LPS-induced Drp1\ntranslocation to nucleus require any post-translational modifications?\nResponse: This is an excellent question. At the moment, no known DRP1 post-translational\nmodification has been reported to drive nuclear translocation. As mentioned by the reviewer, DRP1 is\nextensively regulated by multiple post-translational modifications, including phosphorylation,\nSUMOylation, Ubiquitination, S-nitrosylation, O-GlcNAcylation - making it challenging to pinpoint the\nspecific modification(s) or combination that drive nuclear import. It is also possible that this\ntranslocation is mediated by a mechanism that is independent of DRP1 post-translational\nmodification. Although this is a topic that we fully intend to pursue, it is beyond the scope of the\npresent study. This discussion has been included in the “Limitations of the study” on page 23.\n4. A discussion of the study’s limitations and potential future directions would be valuable. This could\ninclude technical constraints, unanswered mechanistic questions, or border implications for\ntherapeutic strategies for chronic neurodegenerative diseases.\nResponse: The following discussion has been added to page 23: “In addition to some outstanding\nquestions noted elsewhere in this manuscript, this study has several limitations. First, the\nmechanisms responsible for DRP1 nuclear translocation remain unknown. DRP1 undergoes\nnumerous post-translational modifications, including phosphorylation, SUMOylation, ubiquitination, S-\nnitrosylation, and O-GlcNAcylation, making it challenging to pinpoint the specific modification(s) or\ncombination that drive nuclear import. It is also possible that this translocation is mediated by a\nmechanism that is independent of DRP1 post-translational modification. Second, our LPS paradigm\nmodels represent acute early stage of neuroinflammation, it may not fully recapitulate the chronic\ninflammatory milieu of PD. Third, although this study focuses on the role of DRP1-NF-ĸB-lipocalin 2\naxis in mediating neuroinflammation, as a transcriptional factor, DRP1 may regulate additional stress-\nresponsive genes. A broader transcriptional analysis will be necessary to define the full extent of\nDRP1’s nuclear functions.”\nReviewer #2\nIn this paper, Lai et al. presented relevant evidence indicating that DRP1, a protein regulator of\nmitochondrial fission, controls neuroinflammatory response by activating NF-kB. Apparently, this\nmechanism is produced by the protein lipocalin-2 in microglia and astrocytes. These interesting\nstudies propose a novel action of DRP1 on neuroinflammation and link mitochondrial function with\ninflammatory response in the brain. However, several points require clarification before any\npublication process.\nMajor comments\n-The authors declare: “Emerging evidence indicates the central role of neuroinflammation in\nneurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD)2”.\nHowever, this is not emerging evidence, which has been observed and proposed for quite a while.\nThe authors must clarify this information and reduce the enthusiasm of these statements.\nResponse: Thank you for pointing this out. This sentence on page 3 has been modified to “The\ncentral role of chronic neuroinflammation in neurodegenerative diseases such as Alzheimer’s disease\n(AD), Parkinson’s disease (PD) has been well-documented.”\n-The authors must discuss the antioxidant factor NRF2's contribution to NF-KB regulation and its\ninvolvement in the mechanism explored in this manuscript. Several studies suggest that NRF-2\nmodulates the NF-κB response, decreasing inflammatory signaling. If we consider the scope of this\npaper, perhaps NRF2 could be playing a role, or at least this action should be experimentally\ndiscarded.\nResponse: To determine whether NRF2 influences NF-κB activity in our model, we analyzed our\nNanoString dataset and found that Nfe2l2 (the gene encoding NRF2) mRNA levels in ventral\nmidbrains were not significantly changed following LPS treatment as shown in the figure below\n(Supplementary Fig. 2a). We further validated this observation by qPCR, which similarly showed no\nalteration in Nfe2l2 expression (Supplementary Fig. 2b). Together, these results indicate that NRF2\nis unlikely to contribute to the neuroinflammatory response observed in this study. This discussion has\nbeen added to page 7, and the figure below has been included as Supplementary Fig. 2.\nSupplementary Fig. 2. Nfe2/2 levels in LPS-treated mice. Three-month-old Dnm1l+/- and Dnm1l+/+\nlittermates LPS were injected (5 mg/kg, i.p.) or saline control, and the VMB tissue was collected 6h later for\nNanoString nCounter gene expression analysis (a) and qPCR validation (b).\na, Nfe2l2 levels were quantified and normalized to the reference genes Aars, Ccdc127, Cnot10, Tada2b and\nXpnpep1. N=5-8 (2-4F & 3-4M) mice per group, two-way ANOVA.\nb, Nfe2l2 levels, normalized to Gapdh. N=5-7 (2-4F & 3-4M) mice per group, two-way ANOVA followed by\nTukey post-hoc test.\n-Did the authors test Lcn2 levels in any AD mouse models? If not, it is better that the paper focus on\nPD effects related to Lcn2 and not AD.\nResponse: We did not assess the levels of Lcn2 in AD mouse models, but did cite those studies from\nother investigators to support the role of Lcn2 in AD patients and experimental models to illustrate that\nthe role of Lcn2 is not restricted to PD. But as requested, those references and discussions related to\nAD have been removed throughout the manuscript.\n-The authors need to include specific evidence regarding the neurodegenerative role of lipocalin 2 in\nPD.\nResponse: Lipocalin-2 is elevated in the\nsubstantia nigra of patients with PD and in\nMPTP-treated mice. Furthermore, Lcn2-KO mice\nprotect against MPTP-induced\nneurodegeneration, whereas recombinant\nlipocalin-2 injection into the substantia nigra\ninduces marked dopaminergic\nneurodegeneration. In clinical studies,\nRecombinant lipocalin 2 increases α-syn\ncerebrospinal fluid levels of lipocalin-2 in PD\naggregation. A) Stable N27 DA neuronal cells grown\npatients positively correlate with α-synuclein\non glass coverslips were treated with ponasterone A\nlevels and have been proposed as a potential\n(20μM) for 24h to induce α-syn expression, followed\ninflammatory biomarker for PD. However, further\nby purified lipocalin 2 (1μg/ml) for a further 48h. Cells\ninvestigation is required to clarify the were then fixed with 4%PFA, washed and digested\nmechanistic relationship between lipocalin-2 and with proteinase K (PK), and subsequently\nα-synuclein-mediated neurotoxicity. In our other immunostained with Syn1 antibody. Images were\nongoing study, the effects of lipocalin 2 on α- captured using confocal microscopy and PK-resistant\nsynuclein pathology were investigated in the N27 puncta were quantified using Imaris (n=3\nindependent experiments).\nrat dopaminergic neuronal cells with stable\necdysone-inducible expression of human wild-\ntype α-syn. These cells accumulate proteinase-K resistant α-syn after two days of induction by\nponasterone A, an ecdysone analogue. However, lipocalin 2 drastically increased the aggregate\nnumber, indicating the neurotoxic effects of lipocalin 2 in the presence of α-syn. This more detailed\ndiscussion has now been included in the manuscript on page 16.\n-As the authors have investigated DRP1 involvement in neuroinflammatory action, they should be\ntesting the effects on mitochondrial dynamics. They need to show if the change in DRP1 affects the\nexpression of the other mitochondrial dynamic regulators (Opa1, Mfn1/2, Fis1).\nResponse: As suggested, we performed\nqRT-PCR to assess whether reduced DRP1\nexpression in Dnm1l+/- mice would alter the\ntranscription of other key mitochondrial\ndynamics regulators, including Opa1, Mfn2,\nMff, Mief1, Mief2, and Fis1. Our results\n(Supplementary Fig. 11) showed no\nsignificant differences in the expression of\nany of these genes when comparing\nDnm1l+/- mice with their Dnm1l+/+ littermates.\nSupplementary Fig. 11. Levels of mitochondrial\nThese results have been added to\ndynamics regulators in Dnm1l+/- and Dnm1l+/+ mice.\nSupplementary Information. Ventral midbrain tissue of 3-month-old Dnm1l+/- and\nDnm1l+/+ mice littermates were collected for qPCR\n-Also, the authors should specify how DRP1 analysis. Relative gene expressions levels of Opa1, Mfn2,\nactivation induced by several Mff, Mief1, Mief2 and Fis1 were quantified using Gapdh as\nphosphorylation statuses could be involved a reference gene. N=6-7 (2-3F & 4M) mice per\ngroup;unpaired t-tests.\nin the neuroinflammatory regulation induced\nby NF-kB activation. At least two specific phosphorylation residues (616 or 636) can be affected by\noxidative stress or other stressors important in neuroinflammation.\nDRP1 PTMs affected by oxidative stress\nResponse: As noted in our response to Reviewer #1, DRP1 activity can be regulated by multiple\nPTMs, including phosphorylation. In relation to the specific phosphorylation sites highlighted by this\nreviewer, we had previously examined whether phosphorylation at S616 and S637, two well-\ncharacterized PTMs known to be influenced by oxidative stress, might contribute to the results in this\nstudy. Using primary microglia treated with LPS or vehicle control, we isolated nuclear fractions and\nperformed immunoblotting to assess levels of DRP1-pS616 and DRP1-pS637. Our results indicate\nthat neither modification is involved in driving DRP1 translocation to the nucleus. We fully intend to\nfurther investigate the mechanisms underlying DRP1 nuclear import, which may or may not involve\nPTMs.\n-The analysis of mitochondrial dynamics presented in Fig.S3 must be augmented using the macro tool\nfor Fiji-ImageJ MiNA analysis.\nResponse: Please note that we did use this method of mitochondrial analysis as indicated in the\nFigure legend and the Methods section.\nReviewer #3\nLai and colleagues highlight a novel role for dynamin-related protein 1 (DRP1), previously known as a\nkey mediator of mitochondrial fission and autophagy flux, now proposed to act as a transcription factor\nregulating the expression of the Lipocalin 2 gene through the control of Rela (encoding NF-κB). The\nauthors employ a comprehensive set of state-of-the-art\ntechniques, including both constitutive and inducible transgenic mouse models, digital PCR-based quantification of\nproinflammatory mRNA markers in ventral midbrain samples and laser-microdissected brain cells, high-resolution IMARIS\nconfocal imaging, and capillary immunoblotting employing samples from primary microglial cultures.\nAlthough the study supports the identification of a novel function for DRP1 in neuroinflammation, there\nare several concerns that should be addressed prior to publication.\nGeneral Comments:\n1. Although the topic is briefly addressed in the Discussion, the manuscript would benefit from a more\nclearly defined conceptual framework focused on general neuroinflammation in which microglial cells\nare the driving force. Notably, acute intraperitoneal LPS administration does not elicit a brain-specific\nproinflammatory response and astrocytes findings are not explored in depth. The extrapolation of the\nfindings to other neurodegenerative conditions such as Alzheimer’s disease, Parkinson’s disease, or\naging appears premature and should be reconsidered. This is particularly relevant given that the study\nrelies on a single, SNCA model of PD (Dnm1l+/-:SNCA+/- , that is not characterized in the study) and\nincludes only one experiment related to aging. The impact on dopaminergic neuronal survival in\nresponse to LPS is not addressed at all, therefore the relevance in PD models is not analyzed.\nLimiting the scope of the discussion to the specific experimental context would strengthen the\nmanuscript’s coherence and rigor.\nResponse: Our conceptual framework is summarized in Fig. 8, but we agree that the central role of\nmicroglia in this LPS model should be more explicitly stated. To address this, we have revised the Fig.\n8 legend to include the following clarification and supporting references: “Microglia express higher\nbasal levels of TLR4 than astrocytes and therefore respond earlier and more robustly to LPS. In\ncontrast, astrocytic activation requires “priming” by cytokines released from activated microglia,\nmaking microglia the predominant initiators of the inflammatory cascade.”\nAs addressed in an earlier response to Reviewer #2, all discussion regarding the relevance of this\nstudy to Alzheimer’s disease has been removed. The aging data originally presented in Fig. 1, along\nwith the associated discussion, have also been removed. In the revised manuscript, we now provide a\nmore detailed discussion of the relevance of lipocalin-2 to PD (page 16). In addition, our study\nspecifically focuses on dopaminergic neurons, microglia, and astrocytes within the substantia nigra, a\nbrain region directly implicated in PD.\nWe did not assess neurodegeneration in this LPS model because we intentionally selected a\nparadigm that does not produce rapid neuronal loss, thereby avoiding potential confounding effects on\nthe mechanisms under investigation. As discussed above, prior studies using the same LPS regimen\nhave shown that microglial activation emerges within hours of peripheral LPS injection, but\ndopaminergic neurodegeneration in the substantia nigra develops much later, approximately seven\nmonths to observe modest loss (∼23%) and ten months to detect more pronounced degeneration\n(39–47%). Thus, this model reflects an early, pre-degenerative stage of neuroinflammation. We\nbelieve this model represents an early stage of neuroinflammation preceding cell death in PD. This is\ncritical to detect early translocation of DRP1 to the nucleus to initiate neuroinflammation. Additionally,\nwe found that this paradigm markedly increased pro-inflammatory cytokines in the VMB, yet\nmitochondrial respiration in microglia remained intact. This justification and relevant references have\nbeen elaborated more in Discussion (page 16-17).\n2. Please clarify the rationale for using partial instead of full knockout Dnm1l models. In this regard, a\ndeeper characterization of the employed mutant mice with heterozygous Dnm1l knockout is needed.\nFrom the molecular perspective, it would be important to analyze Dnm1l expression levels, both at the\nmRNA and protein level in ventral midbrain extracts, as well as the phosphorylation status of DRP1.\nAdditionally, the expression of key components of the NFκB pathway (such as IKK activation, and the\nprotein levels of IκBα, p50, and p65) should be assessed in comparison to wild-type controls. Related\nto mitochondrial physiology, it would be crucial to describe the impact of deleting one copy of\nDnm1l gene on mitochondrial activity in the brain.\nResponse: Please note that we extensively characterized the constitutive Dnm1l+/- mouse model in\nour previous publication (PMID: 38504290), which was referenced in this study. Briefly, we used\nheterozygous instead of homozygous DRP1-KO mice because complete deletion of Dnm1l is\nembryonically lethal. Quantitative analysis using qPCR and immunoblotting in multiple brain regions\nconfirmed an approximately 50% reduction of DRP1 levels in the Dnm1l+/- mice. These mutant mice\nare indistinguishable from their WT\nlittermates in physical appearance, motor\nand cognitive function. Stereological cell\ncounting shows comparable numbers of DA\nneurons in the substantia nigra pars\ncompacta (SNpc). Assessments of\nmitochondrial morphology and function in\nadult mice show no genotype-dependent\ndifferences. Together, these data indicate\nthat deleting one copy of Dnm1l in the\nheterozygous mice does not negatively\nimpact mitochondria, animal development\nand neuronal viability. However, as\nrequested by this reviewer, we performed\nSupplementary Fig. 10. Differentially expressed\nadditional experiments to characterize this\ngenes (DEGs) in Dnm1l+/- and Dnm1l+/+ mice. RNA was\nmouse model. As seen below, RNAseq\nextracted from ventral midbrain tissue of 3-month-old\ndata show other than Dnm1l, all other Dnm1l+/- and Dnm1l+/+ mice and subjected to RNA\ngenes are not affected in the Dnm1l+/- mice sequencing. The volcano plot displays DEGs between the\n(Supplementary Fig. 10). Consistent with two groups, defined as those showing >1-fold change with\nthese results, no genotypic differences p < 0.05.\nbetween Dnm1l+/- and Dnm1l+/+ littermates were detectable for the following analyses: qPCR for\nmitochondrial dynamics regulators (Supplementary Fig. 11), immunoblotting for phosphorylation of\nDRP1-616 and DRP1-637 - the two major post-translational modifications (Supplementary Fig. 12),\nqPCR for Nfkbia and Rela transcripts as well as immunoblotting for their respective proteins IκBα and\nNFκB-p65 (Supplementary Fig. 13). In combination, these results indicate that Dnm1l+/- mice were\nindistinguishable from their WT littermates. These results have now been included as Supplemental\nInformation and discussed on page 19.\nSupplementary Fig. 12. Protein levels of\nphosphorylated DRP1-S616 and DRP1-S637 in\nDnm1l+/- and Dnm1l+/+ mice. Microdissected Supplementary Fig. 13. mRNA and protein\nventral midbrains from Dnm1l+/+ and Dnm1l+/- levels of Nfkbia/IκBα and Rela/p65 in Dnm1l+/-\nmice was processed for SDS-PAGE and Dnm1l+/+ mice.\nimmunoblotting to assess phosphorylation of\na, NanoString nCounter analysis of Nfkbia and\nDRP1 at Ser616 (a) and Ser637 (c). Interesting,\nRela transcript levels.\npDRP1-S637 migrates faster than pDRP1-S616\nb–c, Immunoblot analysis of IκBα and\nin mouse brain lysates. Total protein staining\ncorresponding quantification.\n(bottom panels) was used as the loading control.\nd–e, Immunoblot analysis of NF-κB p65 and\nb, Quantification of pDRP1-S616 levels from (a).\ncorresponding quantification.\nd, Quantification of pDRP1-S637 levels from (c).\nN = 6–7 mice per group (2–3 F and 3–4 M),\nN = 5–6 mice per group (2–4F and 2–\nunpaired t-tests.\n4M);unpaired t-tests.\nSpecific comments:\nFigure 1. To better illustrate the protective role of Dnm1l, please provide a more comprehensive\ndescription of all dysregulated transcripts comparing LPS-Dnm1l +/- vs LPS-Dnm1l +/+, not just the\ntop ten or the table. Include those validated at the protein level (e.g., IL-6, IL-1β, TNFα) in next\nfigures.\nResponse: A Volcano plot of a total 757\ngenes showing the dysregulated transcripts\ninduced by LPS as shown has been added\nto the revised Fig. 1 in the manuscript. The\nincreases in Lcn2, Cxcl10, and Ccl2 were\nvalidated at protein levels. Interestingly,\nadditional proteins were detectable but not\ntheir transcripts. We believe this is due to\nkey differences between transcript-level\ndetection (Nanostring) and Protein-level\ndetection (MSD multiplex ELISA\nimmunoassay). For example, mRNA can be\ndegraded rapidly while the protein remains\nstable. Nanostring is highly specific but it\nhas a narrow dynamic range of mRNA\nFig. 1a Volcano plot of differentially expressed genes abundance whereas MSD uses\nidentified by NanoString nCounter gene expression electrochemiluminescence and can detect\nanalysis in LPS-treated Dnm1l+/+ versus Dnm1l+/- mice. very low femtogram-levels protein\nconcentrations. Last, there are temporal differences in gene vs protein expression. Changes in mRNA\noften occur earlier and transciently whereas proteins may accumulate or persist longer. This\ndiscussion has now been added to page 7.\nFigure S3. To rule out mitochondrial misbalance is crucial to include positive control (e.g., rotenone or\nparaquat) to validate OCR respiration measures. Also, autophagy should be analyzed, especially\nconsidering previous work by the same group showing that partial Dnm1l knockout improves\nautophagic flux independently of mitochondrial function.\nResponse: Using Supplementary Fig. 4. Mitochondrial\nparaquat as a positive respiration in PQ-treated mouse\nprimary microglia. Cells were treated\ncontrol for\nwith 100 µM PQ for 24 h before\nmitochondrial\nundergoing a mito-stress assay using the\nrespiration, we treated\nXFe96 Extracellular Flux Analyzer.\nthe neuronal N27 cells\na, Representative kinetic traces from the\nwith paraquat, which\nmito-stress assay.\nreduced both basal b, Quantification of oxygen consumption\nand spare respiratory rate (OCR) following PQ treatment.\ncapacity. These N = 3 independent experiments, each with\nresults have now 6–8 technical replicates; unpaired t-tests.\nbeen added to Supplementary Fig. 4 (panels i & j).\nAs requested, we assessed autophagy in LPS-treated primary microglia from Dnm1l+/+ and Dnm1l+/-\npups. Our data show that autophagy impairment induced by LPS was protected by partial loss of\nDRP1 function (Supplementary Fig. 9). This protection is consistent with our previous study. A\ndiscussion of these data has been included on page 9.\nSupplementary Fig. 9. DRP1-KO protects against LPS-induced impairment of autophagy.\nPrimary Dnm1l+/+ and Dnm1l+/- microglia were treated with 100 ng/mL LPS for 1, 3, or 6 h; vehicle-\ntreated cells served as controls. a, SDS-PAGE immunoblot analysis of p62 in whole-cell lysates. Total\nprotein staining (bottom panels) was used as the loading control. b, quantification of p62 levels shown in\n(a). Data represent mean ± SEM from N = 4–5 independent experiments;two-way ANOVA followed by\nTukey’s post hoc test.\nFigure 6. Regarding panel 6e, why are DRP1 nuclear levels similar between Dnm1l+/- and saline-\ntreated controls? There is a reduction only in the cytosolic fraction. This analysis should be repeated\nin whole-cell extracts to assess total protein levels (this is closely related to the second general\ncomment).\nSupplementary Fig. 6. DRP1 levels in\nResponse: As\nmicroglial whole cell lysates.\nmentioned above, we\nPrimary microglia from Dnm1l+/+ and Dnm1l+/-\nquantified the total\nmice were treated with 100 ng/mL LPS for 6 h,\nprotein levels of followed by whole-cell protein extraction using\nDRP1 in the Dnm1l+/- RIPA buffer.\nmice in a previous a, Immunoblot analysis of DRP1 (top panel). Total\nstudy using whole protein staining (bottom panel) served as the\nloading control.\ncell extracts. We\nb, Quantification of DRP1 levels shown in (a).\nconfirmed this\nN = 4 independent experiments; two-way ANOVA\nexpression level in\nfollowed by Tukey’s post hoc test.\nthe present study\nalso using whole cell extracts as suggested by the reviewer. These results have now been added to\nSupplementary Fig. 6. We believe reducing total level of DRP1 by about 50% doesn’t necessarily\ntranslate into reduction of nuclear levels since the correlation might not be linear and only a fraction of\nDRP1 is in the nucleus.\nFigure S4. An explanation\nSupplementary Fig. 5 No\naddressing why Histone H3 differences were detected in\nlevels are reduced in the Histone H3 protein levels.\nnuclear extracts from Dnm1l+/+ and Dnm1l+/- primary\nmutant mice compared to microglia were treated with 100\nwild-type controls could be ng/ml LPS for 6h followed by\nnuclear and cytoplasmic\nadded.\nfractionation. Immunoblotting of\nHistone H3 in the cytoplasmic\nResponse: Thank you for\nand nuclear fractions, and total\npointing this out. To\nproteins per lane were used as\ndetermine whether Histone\nloading control (Left panels).\nH3 levels were truly reduced, The quantification for the levels\nwe increased the sample size of Histone H3 (Right panel).\n(n = 4 instead of the original n\n= 2) and quantified H3 levels in the nuclear fractions. The results show no significant differences\nacross genotypes or treatment conditions. We have now replaced the original plot with a more\nrepresentative one in the revised Supplementary Fig. 5.\nFigures 6 and 7. Since LPS can activate NFκB independently of DRP1, it is crucial to show that the\nidentified DRP1 motif is functional. Therefore, the proposed DRP1 nuclear translocation and binding\nto Rela regulation sites could be strengthened by: Showing p65 protein levels in both cytosol and\nnucleus (Figure 6e–f), highlighting the sequence of the DRP1 motif identified by P7 primers (Figure 7),\ndemonstrating that DRP1 binding leads to increased Rela transcription, to support the claim made in\nline 327.\nResponse: To address the reviewer’s requests, we conducted a series of additional experiments, the\nresults of which are now presented in Fig. 7. First, to further validate DRP1’s transcriptional regulation\nof the Rela promoter, we cloned the 255 bp DRP1-binding region identified by our ChIP assay (Fig.\n7a–b) into the pGL4.20[luc2/Puro] reporter vector (Fig. 7c). Second, we transfected this construct,\nalong with a promoterless control vector, into mouse neuronal cells in the presence of either Dnm1l-\nspecific siRNA or a scrambled control. As shown in Fig. 7d, luciferase activity driven by the DRP1-\nbinding region of the Rela promoter was markedly increased (p < 0.0001), whereas the promoterless\nvector showed no activity. Moreover, Dnm1l siRNA–mediated knockdown significantly reduced\nluciferase activity compared with the scrambled control (p = 0.0048; Fig. 7d). The efficiency of Dnm1l\nknockdown was 71.21±0.68%, as determined by qRT-PCR (Supplementary Fig. 7). These results\ncollectively demonstrate that DRP1 regulates the Rela promoter through a transcriptionally active\nmechanism. Third, we performed a time-course analysis to determine the temporal sequence of\nDRP1 and NF-κB p65 nuclear accumulation following LPS stimulation. As shown in Fig. 7g–i, nuclear\nDRP1 levels increased as early as 0.5 h after LPS exposure, followed by an increase in nuclear NF-\nκB p65 beginning at 3 h, indicating that DRP1 translocates to the nucleus prior to NF-κB p65. Notably,\nwe did not observe a significant increase in cytosolic NF-κB p65 levels up to 6 h after LPS treatment,\nsupporting the notion that DRP1 promotes NF-κB p65 nuclear translocation rather than retention in\nthe cytosol.\nEditorial Note: Figure 7 in this Peer Review File is reproduced with permission from BioRender, Created in\nBioRender. Lai, Y. (2026) https://BioRender.com/m43u985 and Created in BioRender. Lai, Y. (2026)\nhttps://BioRender.com/iii5iv2\nFig. 7. DRP1 enrichment at the Rela promoter upon LPS exposure is transcriptionally active.\na, Schematic of the ~2000 bp Rela promoter region containing seven primer sets (P1–P7) used for ChIP\nanalysis. DRP1 binding was enriched at a ~255 bp region proximal to the Rela transcription start site\nfollowing LPS treatment.\nb, Primary WT microglia were treated with 100 ng/mL LPS or vehicle for 6 h, and ChIP assays were\nperformed to assess DRP1 enrichment at the Rela promoter. “% input” values represent mean ± SEM from\nfour independent experiments, two-way ANOVA with Tukey’s multiple-comparison post hoc test.\nc, Schematic of the pGL4.20[luc2/Puro]-Rela luciferase reporter containing the DRP1-binding region of the\nmouse Rela promoter, along with the promoterless pGL4.20[luc2/Puro] empty vector control.\nd, HT22 neuronal cells were transfected with either the pGL4.20[luc2/Puro]-Rela construct or empty vector for\n24 h, in the presence or absence of Dnm1l siRNA or scramble control. Untransfected cells were included as\nan additional control. Luciferase activity was measured 48 h post-transfection.\ne-i, Primary WT microglia were treated with vehicle or 100 ng/mL LPS for 0.5, 1, 3, or 6 h. Immunoblot\nanalysis of DRP1 (e) and NF-κB p65 (f) was performed in nuclear and cytoplasmic fractions (top panels).\nTotal protein staining (bottom panels) served as loading control. g, quantification of nuclear and cytoplasmic\nDRP1 from (e). h, quantification of nuclear and cytoplasmic NF-κB p65 from (f). i, temporal profiles of DRP1\nand NF-κB p65 enrichment in nuclear and cytosolic fractions demonstrate that DRP1 nuclear translocation\nprecedes that of NF-κB p65. N = 4 independent experiments, one-way ANOVA with Tukey’s post hoc test.\nSupplementary Fig 7. Dnm1l siRNA–mediated\ngene knockdown in HT22 cells. Cells were\ntransfected with either pGL4.20[luc2/Puro] or\npGL4.20[luc2/Puro]-Rela for 24 h in the presence\nor absence of Dnm1l siRNA or a scrambled\ncontrol. Untransfected cells were included as an\nadditional control. RNA was extracted 48 h post-\ntransfection, and Dnm1l mRNA levels were\nquantified using qPCR (normalized to Gapdh).\nData represent mean ± SEM from N = 4\nindependent experiments; one-way ANOVA\nfollowed by Tukey’s post hoc test\nMinor Points:\nLine 38: “Low-grade chronic inflammation” better describes the inflammatory state in\nneurodegenerative diseases.\nResponse: We have changed to “chronic inflammation” as suggested but refrained from stating the\ngrade level of inflammation.\nLine 65: Please elaborate on the neurotoxic role of Lipocalin-2.\nResponse: Please see the response to reviewer #2 above\nLine 71: Use “levels are” instead of “levels is”.\nResponse: Revised as suggested.\nLine 119: Replace “upregulation” with “was upregulated”.\nResponse: Revised as suggested.\nLine 117: Clarify whether the global DRP1-KO mouse refers to a previous study (add reference) or\nunpublished data.\nResponse: A reference has been added.\nResponse: Line 195: The sentence appears to be cut off (please revise).\nResponse: Thank you for pointing this out. This sentence was out of place and has been removed.\nLine 432: The sentence is split across two lines (please correct).\nResponse: This sentence has been corrected.\nLPS (cat# L4391, Sigma-Aldrich): Include the strain type to improve reproducibility.\nResponse: The strain type has been added.\nSupplementary Table 1: The PDF formatting is unclear (please improve the table layout for readability\nmaybe in an Excell file if possible).\nResponse: Excel files have been provided.\nFigure S1a: Add a scale bar to indicate magnification.\nResponse: A scale bar has been added.\nReviewer #4\nLai reports a novel mechanism underlying DRP1-mediated inflammation induced by LPS. Reduced\nDRP1 expression significantly suppresses LPS-induced inflammation, including the proinflammatory\nmolecule lipocalin 2. Upon LPS stimulation, DRP1 translocates from the cytosol to the nucleus, where\nit binds to the promoter region of Rela (encoding NF-κB), activating its gene products and\ndownstream inflammatory cytokines. These findings highlight the important role of proinflammatory\nlipocalin 2 in the brain.\nTogether, this study reveals a previously unrecognized function of DRP1 in mediating\nneuroinflammation via the NF-κB–lipocalin 2 axis. The experiments are well-designed and clearly\npresented. Only minor revisions are suggested.\nSuggestions:\n• Please expand the discussion on the significance and rationale for using the LPS-induced model,\nparticularly in relation to DRP1-mediated inflammation. How well does this model reflect the in vivo\nmechanisms of neuroinflammation?\nResponse: Systemic injection of LPS has been used to induce acute, chronic and progressive\nneuroinflammation resulting in loss of the nigral dopaminergic neurons in animal models of PD. As\naddressed to other reviewers above, this particular LPS regimen was selected because it has been\nreported to induce rapid and sustained microglia activation in the substantia nigra. This is critical to\ndetect early translocation of DRP to the nucleus to initiate neuroinflammation. We believe this model\nrepresents an early stage of neuroinflammation preceding cell death in PD. However, as\nacknowledged in the limitations of this study, the acute nature of this LPS model may not fully capture\nthe chronic inflammatory milieu. This discussion has now been added to page 20.\n• It would also strengthen the manuscript to discuss the outcomes of additional models (if available)\nthat explore the relationship between DRP1-mediated mitochondrial morphology changes,\nneuroinflammation, and neurodegeneration. This could provide further support for the role of DRP1 in\ndisease progression and therapeutic targeting.\nResponse: The following paragraph has been added to the Discussion on page 19: “Given the\ncentral role of DRP1 in cellular stress responses, these findings suggest that DRP1-mediated\ntranscriptional control may contribute to a wide spectrum of inflammatory and neurodegenerative\ndiseases. Indeed, DRP1 inhibition has shown therapeutic potential across numerous models,\nincluding PD10, 11, Alzheimer’s disease12-14, Huntington’s disease12, 15 and amyotrophic lateral\nsclerosis16, where it protects against mitochondrial fragmentation, mitochondrial function,\nneuroinflammation, and neurodegeneration. The present work further expands the protective\nrepertoire of DRP1 inhibition by establishing its transcriptional role in regulating neuroinflammatory\npathways.”\nOnce again, we sincerely appreciate the reviewers’ time, constructive comments and support of this\nstudy. We hope that our responses and revisions satisfactorily address the reviewers’ comments and\nthat the revised manuscript is now suitable for publication in Nature Communications."}, {"round": 2, "reviewer_comments": {"reviewer_1": "Authors sufficiently addressed my concerns.", "reviewer_2": "The authors properly reviewed all comments and included additional data to support their conclusions", "reviewer_3": "NCOMMS-25-49663-T\nThe authors have responded in detail to all the points raised, providing meaningful and relevant experiments that\nsubstantially strengthen the manuscript. In light of these revisions and the improved quality of the study, acceptance of the\nmanuscript would now be appropriate.", "reviewer_4": "I do not have any further concerns. Authors responded very well to my concerns."}, "author_response": ""}]

Decision letter