(2024) 21:123
Li et al. Journal of Neuroinflammation
https://doi.org/10.1186/s12974-024-03125-4

Journal of Neuroinflammation

Open Access

RESEARCH

Deficiency of leucine‑rich repeat kinase 2
aggravates thioacetamide‑induced acute liver
failure and hepatic encephalopathy in mice
Dan Li2,6*†, Shu‑fang Yu2†, Lin Lin1,3,4†, Jie‑ru Guo2†, Si‑mei Huang2, Xi‑lin Wu1,3,5, Han‑lin You1,3,
Xiao‑juan Cheng1,3, Qiu‑yang Zhang1, Yu‑qi Zeng1,3 and Xiao‑dong Pan1,3,4,5,7*Abstract
Background Hepatic encephalopathy (HE) is closely associated with inflammatory responses. However, as a crucial
regulator of the immune and inflammatory responses, the role of leucine-rich repeat kinase 2 (LRRK2) in the patho‑
genesis of HE remains unraveled. Herein, we investigated this issue in thioacetamide (TAA)-induced HE follow‑
ing acute liver failure (ALF).
Methods TAA-induced HE mouse models of LRRK2 wild type (WT), LRRK2 G2019S mutation (Lrrk2G2019S) and LRRK2
knockout (Lrrk2−/−) were established. A battery of neurobehavioral experiments was conducted. The biochemicalindexes and pro-inflammatory cytokines were detected. The prefrontal cortex (PFC), striatum (STR), hippocampus(HIP), and liver were examined by pathology and electron microscopy. The changes of autophagy-lysosomal pathway
and activity of critical Rab GTPases were analyzed.
Results The Lrrk2−/−-HE model reported a significantly lower survival rate than the other two models (24% vs.
48%, respectively, p < 0.05), with no difference found between the WT-HE and Lrrk2G2019S-HE groups. Comparedwith the other groups, after the TAA injection, the Lrrk2−/− group displayed a significant increase in ammonium
and pro-inflammatory cytokines, aggravated hepatic inflammation/necrosis, decreased autophagy, and abnormal
phosphorylation of lysosomal Rab10. All three models reported microglial activation, neuronal loss, disorderedvesicle transmission, and damaged myelin structure. The Lrrk2−/−-HE mice presented no severer neuronal injury
than the other genotypes.
Conclusions LRRK2 deficiency may exacerbate TAA-induced ALF and HE in mice, in which inflammatory responseis evident in the brain and aggravated in the liver. These novel findings indicate a need of sufficient clinical awarenessof the adverse effects of LRRK2 inhibitors on the liver.

†

Dan Li, Shu-fang Yu, Lin Lin and Jie-ru Guo contributed equally to this work.

*Correspondence:
Dan Lidoctorlidan99@fjmu.edu.cn
Xiao‑dong Pan
panxd@fjmu.edu.cn
Full list of author information is available at the end of the article© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to theoriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
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Li et al. Journal of Neuroinflammation(2024) 21:123

Introduction
Hepatic encephalopathy (HE) is a neuropsychiatricabnormality in patients with acute or chronic liver diseases. These patients develop a progressive hypokinetic
rigid syndrome that is termed as “hepatic parkinsonism
or cirrhosis-related parkinsonism”, characterized by progressive ataxia, dystonia, and choreoathetosis accompanied by progressive cognitive dysfunction [1]. Noticeably,
these parkinsonism-like pathophysiologic features canbe manifested in HE, with alterations to the metabolism
and transport of brain dopamine and to receptor integrity at basal ganglia. However, the exact HE pathogenesis
remains obscure.
Of the potential mechanisms underlying HE, thenotion of inflammation-fired neurotoxicity has recently
received growing attention, despite the well-establishedammoniacal poisoning hypothesis. Mounting evidence
highlights the role of microglia in the occurrence anddevelopment of HE-induced central inflammation [2–5].
Studies have documented, in HE patients and rodent
models with liver failure, the activation of microglia andhepatic macrophage, together with increases in proinflammatory factors, including tumor necrosis factor
alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6) [2, 6, 7]. Moreover, Kupffer cells, specialized
liver-resident macrophages, have been found to play animportant role in acute liver failure (ALF) [8]. However,the co-mechanism underlying the activation of microglia
and hepatic macrophages in HE patients remains largely
unelucidated.
The available literature demonstrates that the homeostasis of several tissue-resident macrophage populations, including microglia, is regulated by leucine-richrepeat kinase 2 (LRRK2) [9–11] and that the mutationsof LRRK2 are the commonest genetic cause of Parkinson’s disease (PD) [12–14], with G2019S mutation
documented in sporadic PD [15]. To date, as a complex
GTPase/kinase, LRRK2 orchestrates multiple steps of theendolysosomal pathway and autophagy-lysosomal pathway by interacting with a host of partners and phosphorylating a subset of Rab GTPases [16, 17]. Other studies
have demonstrated that autophagy is implicated in various pathological processes, including HE [18], and thatmodulating autophagy in astrocytes and/or neurons may
represent a novel treatment for liver diseases-associatedHE [19]. Altogether, these findings signify a close involvement of LRRK2 in the HE pathogenesis.
The role of LRRK2 has been explored in various diseases. In inflammatory neurodegenerative disorders,several studies of the central nervous system (CNS) havereported the implication of LRRK2 in various inflammatory modulators such as mitogen-activated protein
kinase (MAPK), nuclear factor of activated T1 cells 1

Page 2 of 19(NFAT1), nuclear factor kappa-B (NF-κB), and regulatorof calcineurin 1 (RCAN1), and its regulation of diverse
inflammatory pathways [20–26]. Cumulative evidence
also suggests that the abnormal activity or mutations of
LRRK2 might prompt the production of pro-inflammatory microglia and that an increase of microglial LRRK2
may promote α-synuclein uptake and clearance [27–30].
Furthermore, some studies have documented an interestingly opposing effect of LRRK2 in CNS and peripheral innate immunity [12]. Still some researchers haveinvestigated the association of LRRK2 expression with
the inflammation in lung and kidney. However, so far,few studies have probed into the role of LRRK2 in liver
inflammation, let alone its role in HE.
In the current study, the role of LRRK2 in the maintenance of liver-brain integrity was investigated in a
TAA-induced HE mouse model. We found that LRRK2
deficiency aggravated the hepatic inflammation of the
LRRK2 knockout mice and partially exacerbated theneuroinflammation. This novel finding signifies a needof sufficient caution in clinical administration of LRRK2
inhibitors.

Methods
Mice

B6.129X1 (FVB)-LRRK2tm1.1Cai/J (stock No. 012453)
and C57BL/6J-Tg (LRRK2*G2019S)2AMjff/J (stock No.
018715) mice were purchased from the Jackson Laboratory (Bar Harbor, Maine, USA). Age-matched, nontransgenic littermates were used as wild-type controls for all
experiments. All animal procedures and experiments
observed the National Guidance for Animal Experimentand were approved by the Animal Ethics Committee of
Fujian Medical University (No. FJMU IACUC 20210313). The mice were allowed free access to food andwater and housed in a standard facility maintained at
22–25 °C on a 12 h/12 h light/dark cycle.
Induction of acute hepatic failure

Three genotypes of mice (aged 6–7 months) were respectively randomized into the treatment group and control
group (45–50 mice/group). The ALF/HE model wasestablished by two doses of single intraperitoneal injection of TAA (200 mg/kg; dissolved in 0.9% sterile saline
beforehand) at an interval of 24 h. Control groups were
injected with 0.9% sterile saline under the same conditions. To prevent hypovolemia, hypokalemia, and hypoglycemia, mice received a solution of 0.45% NaCl, 5%
dextrose, and 0.2% KCl as a substitute for drinking water.
The survival rate of each group was observed every twohours. At 36 h after the first injection, the treatmentgroup underwent the neurological function test, rotarod
test, cylinder experiment, and hindlimb extension test.

Li et al. Journal of Neuroinflammation(2024) 21:123

Mice were anesthetized with a 1.5% ketamine solution
and sacrificed at 48 h after the first injection. The liver,blood, and brain samples were subsequently collected forfurther analysis. The flow chart of the experiment is indicated in Fig. 1A.

Behavioral tests
Neurological function test

The neurological function of mice was assessed with amodified 10-point score scale evaluating the reflexes
and task performance [31]. The former involved startle reflex, grasping reflex, righting reflex, placing reflex,
corneal reflex; the latter included escaping from a circle(1 m in diameter) in less than 1 min, seeking, walking inPage 3 of 19

a straight line, maintaining balance on a beam (1 cm inwidth), climbing onto a square and a round pole. For anabnormal reflex reaction or for each failed task, 1 point
was assigned. Thus, with the brain function score of
normal mice set as 0, a higher score indicated a poorer
neurological function. The neurological function in eachgroup was assessed at 36 h after the first TAA injection.
Rotarod test

This method was similar to a previous study [32]. Mice
were assessed for locomotor coordination and balance using a rotary fatigue apparatus (SA102, Jiangsu
Sans biological technology Co., Ltd.). Before the test,
the mice were placed and trained twice on a rotating

Fig. 1 Survival and behavioral evaluation of mice. A A flow chart for TAA-induced HE mice model. B Survival rate of three TAA-intervened groups(n = 45–50). C Neurological score of each group, with the score of normal mice set as 0 (n = 10–12). D Cylinder test of each group, with the ordinate
showing the percentage of unilateral front paw in the total front paw use (n = 10–12). E Residence time on the rod in the rotarod test in each group(n = 10–12). F The vertical coordinate shows the score of the mice in the hindlimb extension test, with that of the normal mouse set at 4 points(n = 10–12). G The score corresponding to the hindlimb extension of mice in the hindlimb extension test. For neurological score, cylinder test,
rotarod test and hindlimb extension test, data were reported as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, by Kruskal–Wallis testwith Dunn’s comparison

Li et al. Journal of Neuroinflammation(2024) 21:123

rod (30 mm in diameter), which accelerated from 0 to
10 rpm. The training lasted for 15 min, with an intervalof 30 m between the training. Twenty-four hours after
the last training, mice underwent three trials at an interval of 30 m in between, with the rotating rod accelerating from 0 to 40 rpm and lasting for 5 min. The trial wascompleted when an animal fell off or reached a maximum
duration. The time the mouse stayed on the rod in thethree trials was recorded and averaged. A maximum ofsix mice were tested at once and the rotarod was cleaned
after every session.
Hindlimb extension test

As described previously [33], hindlimb extension test was
performed to explore whether the mice suffered fromdystonia. Mice were elevated by the tail by two independent investigators blinded to grouping details. The performance scores ranged from 1 to 4, with a lower scoreindicating severer dystonia. Score 4 was designated as“normal phenotype”, in which the mice reported no dystonia, with legs “V”-shaped widely from the body; score 3
as "mild phenotype", in which mice displayed no dystonia
but were accompanied by trembling or failed to separate
their legs from the body completely; score 2 as "moderate
phenotype", in which mice showed symptoms of dystonia
but were still able to move their legs; score 1 as "severe
phenotype", in which mice failed to demonstrate any leg
extensions or movement. The test score of each mouse
was averaged from two trials, each lasting for 3–5 s.
Cylinder test

The motor dysfunction was evaluated by a method
adapted from a previous study [34]. The animals wereplaced individually inside a transparent cylinder (9 cm
wide and 15 cm high) and observed for 5 min. The number of touching the cylinder wall with the left or rightforelimb or both forelimbs was recorded by an operator
blinded to the experimental groups. The calculation formula was as follows: (L + R)/(L + R + B) × 100% (L = left
forelimb; R = right forelimb; B = both forelimbs), in which
a value of = 50% indicates symmetric use of both forelimbs and that of > 50% signifies right or left forelimb
preference.

Serum liver enzyme and ammonia levels
Mouse blood sample was collected into lithium heparin
anticoagulant tubes by eyeball enucleation. After 10 min
on ice, the sample was centrifuged (3500 rpm, 6 min) andthe upper serum was collected for analyses of aspartate
aminotransferase (AST), alanine aminotransferase (ALT),
and ammonia with a Vitros 5600 automatic biochemical
analyzer (VITROS, USA).

Page 4 of 19

Liver and brain section preparationand histopathological staining
Mice were transcardially perfused with pre-cooled 0.9%
sterile saline and subsequently with pre-cooled 4% paraformaldehyde. Liver and brain samples were isolated
from six groups and post-fixed with 4% paraformaldehyde at 4 °C for 2–4 h. Before the preparation of frozen
liver and brain sections, both samples were cryoprotected
in 30% sucrose at 4 °C for 48 h, with the sucrose refreshedonce after 24 h. Tissues were embedded in OCT compound (Sakura, CA, USA), sectioned (40 μm thick) witha freezing microtome (CM1950, Leica, Germany), andimmersed in PBS at 4 ℃. The tissues were dehydrated
with ethanol, embedded in paraffin, and subsequently
sliced into sections (5 μm thick). The sliced sections wereplaced on poly-l-lysine-coated glass slides (ZSGB-BIO,
Beijing, China).
Liver H&E staining

H&E staining was performed to assess the morphology
and necrosis of liver tissue, and the inflammatory extent.
Briefly, hepatic sections were deparaffinized, rehydrated,and stained with Hematoxylin Staining Solution (ZSGBBIO, Beijing, China) for 2 min. Subsequently, they were
gradiently dehydrated in 75% and 95% ethanol (respectively for 5 min), stained with the eosin staining solution(ZSGB-BIO, Beijing, China) for 2 min, further gradiently dehydrated in 95% and 100% ethanol (respectively
for 5 min), and finally transparentized with xylene twice(2 min per time). The sections were mounted onto coverslips with neutral gum. Cells were viewed under an optical microscope (Leica, Germany).
Liver reticular fiber staining

To assess liver damage in mice, reticular fiber staining was performed on liver tissues. Briefly, liver paraffin sections were deparaffinized and dehydrated andsubsequently treated with 1% potassium permanganate
oxidation liquid (5 min), 2% oxalic acid (1 min), 2% ferric ammonium sulfate (1 min), ammonia silver solution (2 min), 10% formaldehyde solution (2 min), 0.2%
gold chloride solution (1 min), and ponceau-picric acid
(3 min), with a 5-min wash in distilled water between
each step. Finally, the sections were dehydrated, clearedin xylene, and mounted with neutral gum. Staining wasobserved under an optical microscope (Leica, Germany),
revealing a black hue of the reticular fibers.
Brain Nissl staining

Brain sections from the prefrontal cortex, hippocampus(HIP), and striatum were respectively stained with Nissl
staining solution (Beyotime, Shanghai, China) for 10 min

Li et al. Journal of Neuroinflammation(2024) 21:123

following the manufacturer’s instructions. Nissl-positive
cells were visualized under an optical microscope (Leica,
Germany) and analyzed with Fiji software (Fiji, ImageJ
1.53c, NIH, Bethesda, MD, USA).
Immunohistochemical staining

Liver sections were dewaxed with xylene, hydrated
with gradient ethanol, and washed with phosphatebuffer saline (0.01 M PBS). Antigen retrieval was performed by heating sections in a 0.01 M sodium citratebuffer (pH 6.0) or EDTA buffer (pH 8.0). After the treatment with endogenous peroxidase blocker for 20 min toblock endogenous peroxidase activity, the sections wereincubated with serum from the same source of secondary antibody at room temperature (RT) for 20 min toblock nonspecific binding. Tissue sections were incubated overnight with LRRK2 antibody (1: 500, Abcam)
and CD68 antibody (1: 500, Santa Cruz) at 4 °C before
the introduction of secondary antibodies. Sections were
further incubated in DAB to produce a brown reaction
product, counterstained with hematoxylin, dehydrated,
and transparentized. Similarly, brain sections received
immunohistochemical staining with Iba1 antibody(1: 1000, WAKO), but without dewaxing, hydration, andantigen retrieval. Staining was observed under an optical microscope (Leica, Germany) and evaluated with Fiji
software (Fiji, ImageJ 1.53c, NIH, Bethesda, MD, USA).
Immunofluorescence staining

The expression of LAMP1 and Rab10 in liver tissues wasdetermined by immunofluorescence staining. Briefly, the
40-μm liver sections were incubated in glycine solution
at RT for 20 min after PBS washes. After the treatment
with a blocking solution (0.3% Triton X-100, 1% BSA
and 10% donkey serum) at RT for 1 h, the sections wereincubated with primary antibodies (LAMP1, Rab10) at
4 °C overnight and subsequently with donkey anti-mouse
IgG H + L (Alexa ­Fluor® 488), donkey anti-rat IgG H + L(Alexa Fluor® 594) at RT for 1 h. To remove the autofluorescence in liver tissues, the sections were treated witha Vector True VIEW Autofluorescence Quenching kit(Vector, CA, USA) at RT in the dark for 2 min. Then, thenuclei were counterstained with DAPI (1: 10,000, Thermo
Fisher).
The expression of TNF-α and IL-6 in PFC tissues wasdetermined by immunofluorescence staining. The 40-μm
brain sections were incubated in glycine solution at RT
for 20 min after PBS washes. After the treatment with ablocking solution (0.3% Triton X-100, 1% BSA and 10%
donkey serum) at RT for 1 h, the sections were incubated with primary antibodies (TNF-α, Proteintech; IL-6,
CST) at 4 °C overnight and subsequently with donkey
anti-mouse IgG H + L (Alexa ­Fluor® 488) and donkey

Page 5 of 19

anti-rabbit IgG H + L (Alexa F­ luor® 594) for TNF-α, donkey anti-rabbit IgG H + L (Alexa F­ luor® 488) and donkey
anti-goat IgG H + L (Alexa F­ luor® 594) for IL-6 at RT
for 1 h. Then, the nuclei were counterstained with DAPI
(1: 10,000, Thermo Fisher).
Finally, the anti-fluorescence quencher was used to seal
the sections. The images were obtained by immunofluorescent microscopy (Observer A1, Zeiss, Germany) andphotographed under an inverted fluorescence microscope (Leica, Germany).
Image analysis and quantification of Nissl staining, Iba1
staining and immunofluorescence staining

After the relevant experiments, images were collected using identical image acquisition parametersand processed uniformly. For quantification, images
were imported to Fiji software (Fiji, ImageJ 1.53c, NIH,
Bethesda, MD, USA) and converted into 8-bit images.
The percentage of the area occupied by cells was determined with the polygon tool; the thickness of different
brain regions with straight-line tool; and counting withthe cell counter tool. To analyze the number and morphology of neurons in the brain tissue, Nissl-stained cells
were counted and the percentage of cells and thickness ofdifferent hippocampal regions were calculated from random visual fields. For quantification of microglial cells,
Iba1 + cells were counted per high-power field (HPF).
Sholl analyses were performed to quantify the degreeof microglial activation. Images were processed by setting densitometric thresholds, smoothed, binarized, andskeletonized using the Skeletonize Plugin [35]. Templatesof concentric circles were overlaid onto the center of adigitized cell soma, increasing the radius by a length of
3.0 μm to the end of the branch. The total number ofover-threshold branches that intersected each circle
was calculated and compared among groups. To detect
the expression of specific markers in the liver tissues,
the fluorescence intensity was quantified and expressed
as mean fluorescence intensity (MFI). All images were
processed and quantified by independent investigators
blinded to the experimental condition.

Western blotting
The liver tissues were lysed for 30 min in a RIPA buffer(Beyotime, Shanghai, China) containing a phosphataseinhibitor and a protease inhibitor (Roche). Subsequently,
the tissue lysates were centrifuged at 4 °C for 15 min at
14,000 rpm to collect the supernatant, which was quantified by the BCA method (Beyotime, Shanghai, China).
Protein extract (40 μg/well) was loaded into the ten
wells in the 12% and 8% SDS–polyacrylamide gel (SDSPAGE gel) and transferred to nitrocellulose membranes(NC membranes). Bands were blocked with 5% skimLi et al. Journal of Neuroinflammation(2024) 21:123

milk/TBST for 2 h and incubated in primary antibodies at 4 °C overnight, and further incubated in secondary antibodies at RT overnight. Bands were visualizedwith Bio-Rad ChemiDoc Imaging System using BeyoECL
Star (Beyotime, Shanghai, China). Densitometric analysis was performed with Fiji software (Fiji, ImageJ 1.53c,
NIH, Bethesda, MD, USA) and the protein band intensity
was calculated by normalizing against the GAPDH band
intensity. LC3B-II/LC3B-I ratio was calculated to evaluate the autophagy level of liver tissues in each group.

RNA isolation and RT‑qPCR
RNA was extracted from the liver tissues with a TRIzol
Reagent (Invitrogen). cDNA synthesis was performed
using a Revert Aid First Strand cDNA Synthesis Kit following the manufacturer’s instructions (Thermo Science,
Waltham, USA). The quantitative RT-PCR (RT-qPCR)
amplification was performed using FastStart Universal
SYBR Green Master (ROX) (Roche) on the 7500 RealTime PCR system (Applied Biosystems, South San Francisco, CA, USA). GAPDH mRNA levels were used as aninternal normalization control. The specific primers are
specified in Supplementary Table 1.
TEM specimen preparation, image analysis,and quantification
Liver, prefrontal cortex, and striatum were quickly isolated from anaesthetized mice. Tissue blocks (1 ­mm3)
were cut immediately and fixed with 3% glutaraldehyde-1.5% paraformaldehyde at 4 °C for 24 h; the tissues
were post-fixed with 1% osmium acid-1.5% potassium
ferrocyanide at RT for 1.5 h; they were then rinsed with
PBS, stained with 70% uranium acetate saturated alcohol solution, dehydrated with alcohol-acetone, andembedded in epoxy resin (618 embedding medium).
The ultrathin sections (90 nm) were stained with uranyl
acetate and lead citrate for 5–15 min, and photographed
under a TECNA1 transmission electron microscope (FEI,TECNAI G2). The myelinated and unmyelinated fibers
were counted to calculate the percentage of myelinatedfibers. The G-ratio of myelinated fibers was calculated
as the ratio of the axonal diameter to the diameter of the
myelinated fiber with Fiji software (Fiji, ImageJ 1.53c,
NIH, Bethesda, MD, USA). The myelinated fibers shorter
than 1 µm were referred to as the small myelinated fibers.
Liver lysosome isolation
Crude lysosomal fractions were isolated from the livertissues using a Lysosome Isolation Kit (No. LYSISO1,
Sigma-Aldrich) following the manufacturer’s instructions. Briefly, an appropriate amount of liver tissues wasweighed and washed with PBS and subsequently mincedwith 1X Extraction. The homogenate was centrifuged atPage 6 of 19

1000g for 10 min at 4 ℃ to remove large particles. The
above steps were repeated and the supernatants were collected and centrifuged at 20,000g for 20 min. The pellets
were saved and resuspended in 1X Extraction to obtaincrude lysosomal fractions.

Enzyme linked immunosorbent assay
All reagents were prepared following the instructions of
the assay kits (EK282HS, EK206HS, EK296, EK201BHS,
EK280HS, Liankebio). The brain tissues samples anddiluted serum were added into the plates before a 90-min
incubation. After washing, the plates received antibody, streptavidin labeled with horseradish peroxidase,enhancement solution, and streptavidin labeled withhorseradish peroxidase, in that order, and respectively
incubated for 30 min, 30 min, 15 min, and 15 min. Plates
were washed each time before addition. The tetramethyl
benzidine was incubated in the dark for 5–30 min. The
stop solution was added into the plates. Lastly, the opticaldensity was detected at 450 nm and 570 nm. The quantification of the IL-1β, IL-6, IL-8, TNF-α, and IFN-γ concentrations was performed by calibration with a standard
curve.
Statistical analyses
Survival analysis was presented as Kaplan–Meier survival
curves with log-rank statistics. Data were expressed asmean ± SEM. For homogeneous variance, the data wereanalyzed by one-way analysis of variance (ANOVA) withpost hoc Tukey multiple comparison test; for nonhomogeneous variance, the data were compared by non-parametric Kruskal–Wallis rank sum test with post hoc Dunn
multiple comparison test. All statistical analyses were
performed with GraphPad Prism V.7.0 (GraphPad Prism
Software, San Diego, California, USA). A value of p < 0.05
was considered statistically significant.
Results
LRRK2 deficiency exacerbates neurological dysfunction
and increases mortality in TAA‑HE mice

Mouse models of TAA-HE were established to study therole of LRRK2 (Fig. 1A). After the TAA intervention, themice displayed an acute response: flagging spirit, sluggish
and progressively-weakened response to external stimuli,
clammy skin, yellowish urine, and impaired motor ability(Fig. 1B). The TAA-treated groups reported significant
differences in survival rate: 48% in the WT-HE group,
46.5% in the Lrrk2G2019S-HE group, but only 25.4% in
the Lrrk2−/−-HE group (Fig. 1B) (Pairwise comparison
between groups shown in Supplementary Fig. 1), indicating that LRRK2 deficiency significantly increases themortality of mice after TAA intervention.

Li et al. Journal of Neuroinflammation(2024) 21:123

The behavioral tests revealed that TAA intervention
greatly increased the brain function score of mice, with
Lrrk2−/−-HE mice scoring the highest (Fig. 1C). TAAtreated mice reported motor incoordination (Fig. 1D),
motor fatigue (Fig. 1E), and dystonia (Fig. 1F, G). Interestingly, the mice of the two genotypes exhibited different behavioral performances after drug administration.
The Lrrk2−/−-HE mice displayed the most serious
impairment in the cylinder test and hindlimb extension
test (p < 0.05), while Lrrk2G2019S-HE group performed
the worst in the rotarod test (p < 0.05). Overall, theresults indicate that LRRK2 deficit impacts the TAAHE mice more adversely and exacerbates the survival ofPage 7 of 19

the TAA-HE model when compared with an aberrant
LRRK2 overexpression.
LRRK2 deficiency aggravates liver injury and neuronal loss
by inducing peripheral and central inflammation in TAA‑HE
mice

The impact of LRRK2 on the function of important target organs was investigated by assessing pathologicalchanges of liver and brain in the TAA-HE mice. Macroscopically, compared with the control group, the liver of
the TAA-intervened mice featured general congestion,a swollen, dark red, rough surface, and extensive necrosis, especially in the Lrrk2−/−-HE mice (Fig. 2A), whereas

Fig. 2 Pathological and physiological changes of liver. A General view of liver in each group. B Absolute and relative liver weight of each group(n = 10–12). C Absolute and relative brain tissue weight of each group (n = 10–12). D Pathological changes of liver tissue of each group by H&E
histochemistry (Scale bars: 50 μm). E Reticular fiber changes of liver tissue of each group by reticular fiber staining (Scale bars: 50 μm). F Livertissue of each group by transmission electron microscopy (Scale bars: 1 μm). G Levels of blood ammonia, ALT, and AST of each group (n = 10–12).
For absolute liver weight, relative liver weight, absolute brain tissue weight, relative brain tissue weigh, ammonia, ALT and AST, data were reported
as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, by Kruskal–Wallis test with Dunn’s comparison

Li et al. Journal of Neuroinflammation(2024) 21:123

no significant change was evident in the brain. The livers, brains and bodies of the mice were weighed and the
proportion of liver and brain weight to that of the wholebody was respectively calculated. The results showedthat absolute and relative liver weight in TAA-intervened groups markedly increased (p < 0.05, respectively)(Fig. 2B). Notably, among all TAA-intervened groups,
the absolute and relative liver weigh increased most significantly in the Lrrk2−/−-HE mice (p < 0.05); however, nosignificant difference was found in the absolute or relative weight of brain tissues (Fig. 2C). In H&E staining,
compared with the control group, the TAA-intervened
groups displayed hepatocytic edema and ballooning,
reticular fiber collapse, infiltration of inflammatory cells(mainly macrophages and neutrophils), and punctate
or focal necrosis of the hepatic lobules centered on thecentral vein, with the most significant inflammatorychanges in the liver of the Lrrk2−/−-HE mice (Fig. 2D).
Reticular fiber staining showed that the reticular fibers were complete in the control group, disconnected in
the WT-HE and Lrrk2G2019S-HE groups, and collapsed in
the Lrrk2−/−-HE group (Fig. 2E). The transmission electron microscopy revealed edema and steatosis of variable
degrees in liver cells from the TAA-intervened mice, withthe most serious hepatocytic necrosis in the Lrrk2−/−-HE
mice (Fig. 2F). Nissl staining showed the pathological damage in brain tissues after the TAA treatment(Fig. 3A–D and Supplementary Fig. 2). Layer V neurons
were consistently selected from the PFC, revealing swollen and fragmented neurons after the TAA intervention (Fig. 3C). Meanwhile, after the TAA treatment, theLrrk2−/− mice showed exacerbated partial neuronal loss,
which was not the worst when compared with that ofother TAA-intervened groups, while the Lrrk2G2019S micereported no significant changes in the PFC, STR and HIP
before or after the TAA intervention (Fig. 3E, F).
Besides pathological manifestations, biochemical differences were evident in the liver. The TAA intervention
caused serum hyperammonemia accompanied by significant increases in aspartate aminotransferase (AST)
and alanine aminotransferase (ALT) (Fig. 2G). Likewise,
the Lrrk2−/−-HE mice reported a higher level of bloodammonia (p < 0.05). The above results indicate that acute
HE mice of different genotypes feature acute liver inflammation and necrosis to varying degrees, which can beexacerbated by LRRK2 deficiency, implying that LRRK2
plays a dual distinct role in hepatic inflammatory injury
in HE mice.
LRRK2 deficiency aggravates myelin damage and vesicletransport disorders in TAA‑HE mice

Next, we analyzed the nerve fibers by transmission
electron microscopy. The results showed disrupted

Page 8 of 19

myelin sheath, hyperdense axoplasm, and vacuolizationin the HE mice, particularly in the Lrrk2G2019S-HE mice
(Fig. 4A). G-ratio analysis revealed that the TAA intervention led to demyelination and regeneration of neural fibers in the STR and PFC regions, thus resulting in
an increased G-ratio (Fig. 4B). TAA intervention also
reduced the proportion of myelinated neural fibers in the
STR (Fig. 4C). After the TAA intervention, the differences
observed were mainly evident in the STR, with a significantly higher overall fiber G-ratio in the Lrrk2G2019S-HE
mice than in the WT-HE and Lrrk2−/−-HE groups. Thisphenomenon was particularly noticeable in the small
myelinated fibers of the Lrrk2G2019S-HE mice (Fig. 4B),
accompanied by a decrease in the proportion of small
myelinated fibers (Fig. 4C). These results suggest that
LRRK2 overexpression aggravates myelin damage in the
TAA-HE mice.
The functional blockade of nerve conduction was further explored. The PFC brain sections of the mice were
examined by transmission electron microscopy, reporting a similar number of asymmetric and symmetric synapses in each group (Fig. 4D, E). In asymmetric synapses,the vesicle size in the Lrrk2−/−mice was not consistentand distributed sparsely, with some vesicles exhibitinga significantly greater diameter (Fig. 4D, asterisk in ii),
which was prevalent in all groups after the TAA injection (Fig. 4D, iv–vi); in symmetric synapses, the vesicles
were more sparsely-distributed in the Lrrk2−/− micethan in the other groups (Fig. 4E, i–iii) and all HE groups
reported abnormal vesicle shape (Fig. 4E, iv–vi). Of note,in asymmetric synapses, the Lrrk2−/−-HE group exhibited more pronounced abnormalities in vesicle destruction than the WT-HE and Lrrk2G2019S-HE groups. Apartfrom the varied sizes, the vesicle of the Lrrk2−/−-HE micereported an overall structural disorder, including unusually-formed endocytic intermediates (Fig. 4D, asterisk inv) and protein-coated vesicles with less density (Fig. 4D,v). These findings suggest that LRRK2 knockout aggravates the injury to the vesicle shape in the TAA-HE mice.
Effect of LRRK2 knockout on the changes of acute phasecytokines in the liver and nervous system of TAA‑HE mice

We further explored the role of LRRK2 in the occurrence and development of acute liver inflammation. First,
the expression of LRRK2 was verified in each group by
western blotting and immunohistochemical staining.
The result reported a significantly higher LRRK2 expression in the Lrrk2G2019S mice than in the WT mice. Afterthe TAA intervention, LRRK2 was upregulated in boththe Lrrk2G2019S mice and WT mice. CD68 and CD45,
two molecular markers on the surface of macrophages,
were detected by western blotting and immunohistochemical staining. Similarly, the expression of CD68 andLi et al. Journal of Neuroinflammation(2024) 21:123

Page 9 of 19

Fig. 3 Representative images showing Nissl staining in brain tissues and quantification of damaged neurons. A Low-magnification imagesof the PFC (as shown in B) (Scale bars: 100 μm). Dashed lines indicate the borders of layer V. C High-magnification images of neuronal morphology
in layer V of the PFC as indicated by area with dashed lines in A (Scale bars: 5 μm). D Low-magnification images of the hippocampal DG regions(Scale bars: 100 μm). E Quantification of the layer V neurons from the PFC, the dorsal and ventral STR (n = 6–8, 30–55 fields of views were observedfor each mouse). Data are represented as mean ± SEM. and analyzed by one-way ANOVA test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).
F Thickness of the cell layer in the hippocampal CA1, CA3 and DG (n = 6–8, 55–85 fields of views were observed for each mouse). Data arerepresented as mean ± SEM. and analyzed by one-way ANOVA test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

CD45 increased after the TAA intervention, with the
Lrrk2−/−-HE mice reporting the highest CD68, followedby the Lrrk2G2019S-HE mice, and then the WT-HE mice(p < 0.05) (Fig. 5A–C). These results suggest that after the

TAA intervention, LRRK2 knockout promotes the activation and recruitment of macrophages in the liver, especially in the portal area of the Lrrk2−/−-HE mice.

Li et al. Journal of Neuroinflammation(2024) 21:123

Moreover, the mRNA levels of CD86 and acute inflammatory cytokines such as IL-1β, IL-6, IL-8, TNF-α, IFN-γ
significantly increased in all TAA-intervened mice, especially in the Lrrk2−/−-HE mice (p < 0.05) (Fig. 5D and
Supplementary Fig. 3A). In the plasma, ELISA analysis
reported an upregulation of the inflammatory factors
TNF-α and IL-6 in the TAA-intervened mice, especiallyin the Lrrk2−/−-HE mice (Fig. 5E). These results evidence
the severest peripheral inflammation in the Lrrk2−/−-HE
mice, consistent with the inflammatory response in theliver.
In the brain tissues of the Lrrk2−/−-HE mice, qPCR
results showed that acute inflammatory cytokines such as
IL-1β, IL-6, IL-8, TNF-α, IFN-γ were upregulated (Fig. 5F
and Supplementary Fig. 3B), consistent with those of liverand peripheral circulation. Of note, the baseline values ofsome inflammatory factors in the Lrrk2G2019S mice werehigher than those of the other two control groups. Afterthe TAA intervention, the proinflammatory cytokines
declined in the Lrrk2G2019S mice but no significant difference was found when the Lrrk2G2019S-HE mice were,respectively compared with its own control group andthe WT group. Additionally, the ELISA analysis of the
PFC tissue homogenates indicated that compared withthe other genotype counterparts, Lrrk2−/− mice reported
a higher level of increased cytokines after the TAA intervention (Fig. 5G). In the Lrrk2−/−-HE mice, the immunofluorescence staining revealed that TNF-α and IL-6 werereleased from the microglia to the extracellular space andinteracted with other cellular entities, including neurons
with large nuclei (Fig. 5H). The above results indicate theinvolvement of LRRK2 in ALF and HE by promoting theactivation and recruitment of macrophages to the liver
and the release of inflammatory cytokines.
LRRK2 deficiency increases microglial dysfunction
in TAA‑HE mice

Given the role of microglial activation in HE progression,
we next explored microglial activation in acute cerebral
inflammation. With Iba1 as a general microglial marker,
the microglial activity was detected by examining thePage 10 of 19

microglial morphological changes in the dorsal STR and
PFC (Fig. 6A). The results showed that after the TAA
intervention, microglia were activated in all TAA-intervened mice, with the overall density and branch numberof Iba1-positive microglia significantly increased, andmicroglial ramification retracted and thickened (Fig. 6B–
D). Compared with the control groups, the Lrrk2−/−-HE
and Lrrk2G2019S-HE mice reported no significant change
in the fraction of the total area. However, the Lrrk2G2019SHE mice reported retracted and thickened microglial
ramifications in the Sholl analysis (Fig. 6B, D) and anincreased number of microglia in the PFC (Fig. 6C). Afterthe TAA intervention, the analysis of the phenotypesof microglia in each genotype revealed a significantly
higher number of glial cells in the PFC of the Lrrk2G2019SHE mice than in that of the Lrrk2−/−-HE group in termsof absolute cell count (Fig. 6C). Sholl analysis further
showed that compared with the WT-HE counterparts,
the Lrrk2−/−-HE mice exhibited a slightly denser andelongated branching of microglia in the PFC and STR. In
contrast, the Lrrk2G2019S-HE mice indicated a contraction
and thickening of glial cell branches (Fig. 6B, D). These
results evidence that microglia exert distinct effects
on the morphological changes in the Lrrk2−/−-HE andLrrk2G2019S-HE mice.
LRRK2 deficiency interferes with Rab10 phosphorylationand the autophagic‑lysosomal pathway in TAA‑HE mice

Since autophagy plays an important role in the progressof inflammation caused by LRRK2-related diseases, we
examined the expressions of three important autophagyrelated proteins, Beclin 1, microtubule-associated protein
1 light chain 3 beta (LC3B), and p62 to investigate thechanges of autophagy in mice undergoing acute inflammation after the TAA intervention. Western blotting
revealed significantly up-regulated protein levels of LC3B
I-converted LC3B II and p62 in the HE mice when compared with the controls, despite no significant change in
Beclin 1 protein level (Fig. 7A and Supplementary Fig. 4).
This finding suggests that despite the initial activation of
autophagy of hepatocytes during the acute inflammation,(See figure on next page.)
Fig. 4 The morphological characterization of myelinated axons by electron microscopy in brain tissues and quantification of the regenerationand distribution of myelin sheaths. A Representative images of myelinated axons at STR by electron microscopy (Scale bars: 1 μm). B Quantificationof myelin g-ratios at PFC and STR (n = 3, 5–10 fields of views were observed for each mouse). Data are represented as mean ± SEM and analyzed
by one-way ANOVA test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). C Ratio of myelinated and unmyelinated fibers in STR (n = 3); ratio of large
and small myelinated fibers in STR (n = 3) (Chi-square test; *p < 0.05, **p < 0.01). D, i–vi The images of vesicle at asymmetric synapses by electron
microscopy (Scale bars: 500 μm). (ii) The vesicle of Lrrk2−/− mice exhibited inconsistent size and sparse distribution, some with a significantly greater
diameter (asterisk in ii). (v) The overall structural disorder in vesicle of the Lrrk2−/−-HE mice, including unusually formed endocytic intermediates(asterisk in v) and protein-coated vesicles with obscure borderline. E, i–vi The images of vesicle at symmetric synapses by electron microscopy (Scalebars: 500 μm). (i–iii) The sparser vesicle distribution in the Lrrk2−/− mice (ii) than in other groups. (iv–vi) The abnormal vesicle shape in all TAA-HE
groupsLi et al. Journal of Neuroinflammation

Fig. 4 (See legend on previous page.)(2024) 21:123

Page 11 of 19

Li et al. Journal of Neuroinflammation(2024) 21:123

Page 12 of 19

Fig. 5 The expression of inflammatory factors in peripheral circulation, liver, and brain tissues of each group. A Liver proteins CD68 and LRRK2
expressions were assessed in each group by immunohistochemistry. B Liver total proteins LRRK2, CD68 and CD45 expression in each group (C)quantified by densitometry (n = 6). D The liver mRNA levels of CD86, IL-1β, IL-6, IL-8, TNF-α and IFN-γ, were assessed by qPCR (n = 6). E Peripheral
IL-6 and TNF-α levels were detected by ELISA (n = 10–12). F LRRK2, IL-1β, IL-6, IL-8, TNF-α and IFN-γ mRNA expression in the PFC of each group wereassessed by qPCR (n = 6). G IL-1β, IL-6, IL-8, TNF-α and IFN-γ levels in the PFC of each group were detected by ELISA (n = 6). H The colocalizationof Iba1-expressing microglia (red) and TNF-α/IL-6 (green) in each group. DAPI (blue) was used as a nuclear stain (Scale bars: 25 μm). Representative
fluorescent signal intensity plots (arrows in colocalization images) in each genotype after TAA intervention are indicated below the images.
For protein, ELISA and mRNA analyses, data are reported as mean ± SEM. and analyzed by Kruskal–Wallis test with Dunn’s comparison. *p < 0.05;**p < 0.01; ***p < 0.001; ****p < 0.0001(See figure on next page.)
Fig. 6 The representative images and quantification of Iba1-expressing microglia in brain tissues. A Low-magnification images of the PFC
and dorsal STR (Scale bars: 500 μm). B High-magnification images of the PFC and the dorsal STR, with representative images of binarized microglial
morphology (Scale bars: 50 μm). C Cell counts and calculation of area fraction of microglia in the PFC and dorsal STR (n = 6–8, 30 fields of views
were observed for each mouse). Data are represented as mean ± SEM. and analyzed by one-way ANOVA test (*p < 0.05, **p < 0.01, ***p < 0.001,****p < 0.0001). D Sholl analyses of microglia morphology (n = 6–8, 10 fields of views were observed for each mouse)

autophagy flow is obstructed in the TAA-HE modeland fails to maintain the homeostasis of the internal
environment.
Lysosome is the end point of numerous vesicle trafficking pathways, including autophagic and endocytic

lysosomal pathways. Western blotting was performed todetect the levels of the lysosome-associated membraneproteins 1 (LAMP1) and 2 (LAMP2), and the soluble lysosomal enzyme Cathepsin D (Cath-D), the latter acting
as an indirect marker enzyme to evaluate the lysosomal

Li et al. Journal of Neuroinflammation

Fig. 6 (See legend on previous page.)(2024) 21:123

Page 13 of 19

Li et al. Journal of Neuroinflammation(2024) 21:123

Page 14 of 19

Fig. 7 The expression of autophagy-related genes in each group. A Liver lysosomal proteins (LRRK2, CD45, CD68, LAMP1, LAMP2, Cath-D, Beclin1,
LC3B, p62, and Rab10) in the liver tissues of each group were examined by western blotting (n = 6). B The lysosomes (red arrows), autophagosomes(yellow arrows) and autolysosome (orange arrows) of each group were shown by transmission electron microscopy (Scale bars: 800 nm). C The
colocalization of Lamp1 (red) and Rab10 (green) in each group. DAPI (blue) was used as a nuclear stain (Scale bars: 50 μm). For protein analyses, data
are reported as mean ± SEM. and analyzed by Kruskal–Wallis test with Dunn’s comparison. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

function. In the acidic milieu of lysosomes, pro-Cath-D
can be processed into intermediate active Cath-D andmature Cath-D. The results reported a markedly increasein LAMP1, LAMP2, and pro-Cath-D, but a decrease inintermediate active Cath-D and mature Cath-D in the
TAA-HE mice (Fig. 7A and Supplementary Fig. 4). Compared with the WT and Lrrk2G2019S mice, the Lrrk2−/−-HE mice reported the highest expression of pro-Cath-D.
These findings suggest that LRRK2 deficiency aggravates
the TAA-induced blockage of autophagic-lysosomal
pathway during the acute inflammatory response. The

electron microscopy displayed the changes in the number of secondary lysosomes and autophagolysosomes in
the liver after the TAA intervention, as well as the swelling and degeneration of cells and organelles (Fig. 7B).
In the Lrrk2−/−-HE mice, an increase in autophagosomal formation and lysosomal destruction was observed.
These results suggest that LRRK2 deficiency impairs the
progress of autophagic-lysosomal pathway in the liver
during the acute inflammatory response.
As LRRK2 can be recruited to stressed lysosomes,
where it recruits and phosphorylates its substrate, smallLi et al. Journal of Neuroinflammation(2024) 21:123

Rab GTPases, to maintain lysosomal homeostasis [36],we next investigated if LRRK2 affects lysosomal homeostasis through Rab protein in the TAA-HE model. Lysosomal proteins were purified with a lysosome isolation
kit. The results showed that after the TAA intervention
no significant difference was found in the expressionof Rab5, Rab7, Rab8 and Rab29 in the liver lysosomes
among groups (Supplementary Fig. 5) and that the total
Rab10 was increased in all TAA-intervened mice andsignificantly upregulated in the Lrrk2G2019S-HE mice andLrrk2−/−-HE mice (Fig. 7A). Subcellular localization of
Rab10 and LAMP1 was detected by immunofluorescence(Fig. 7C). As expected, the WT and Lrrk2G2019S mice,
after the TAA intervention, reported an increased protein level of Rab10 and enhanced respective colocalization with lysosomes, while the Lrrk2−/− mice reported anincrease in Rab10 protein level and an increased colocalization of LAMP1 with lysosomes. As Rab10 can be phosphorylated at Thr73 by LRRK2, the phosphorylation of
Rab10 was detected, which was lower in the Lrrk2−/−-HE
mice than in the WT-HE or Lrrk2G2019S-HE mice (Fig. 7A
and Supplementary Fig. 4). These results suggest that in
the TAA-HE mice, LRRK2 may interfere with the lysosomal homeostasis by regulating Rab10 phosphorylation,
thereby affecting autophagy.

Discussion
Currently, the pathogenesis of HE, an important complication of end-stage liver disease, is poorly elucidated andmuch remains to be desired, despite the intense research
into the role of hyperammonemia in its pathogenesis.
Recently, the notion of the systemic inflammation theory
has attracted growing attention, which hypothesizes thatthe inflammatory response may promote the occurrence
and pathophysiological progression of HE.
The current study capitalized on the strong association between LRRK2 and systemic inflammation andexplored the pathogenesis of TAA-induced ALF and HE.
Our data first revealed that in TAA-induced mouse models of ALF and HE, the survival rate was twofold higher
in the control group than in the Lrrk2−/−-mice and that
LRRK2 deficiency significantly aggravated TAA-induced
acute hepatic inflammation, which featured, to varying degrees, large focal hepatocytic necrosis, abnormal
morphology in residual hepatocytes, edema, hepatocytic degeneration, cholestasis, collapse of reticularfiber scaffolds, infiltration of lobular inflammatory cells,
and significant increase of acute inflammatory factors
in tissues and peripheral circulation. Further mechanistic research reported that LRRK2 knockout affected the
autophagy-regulated cell homeostasis to protect against
inflammation by interfering with hepatic autophagiclysosomal signaling pathway. However, in the central

Page 15 of 19

nervous system, despite the disrupted synaptic vesicle
trafficking, LRRK2 deletion, to some extent, attenuatedthe myelination and neuronal loss via microglial activation-mediated central inflammation. These findings
suggest that LRRK2 affects the central and peripheral
systems in distinct inflammatory regulatory mechanisms.
This discrepancy indicates a potential immune exemption
and protection mechanism in the central nervous system,
signifying that LRRK2 may fundamentally regulate thesystemic immune inflammation in the TAA-HE modelthrough peripheral regulation and that the observed highmortality rate of the model may be attributed to peripheral rather than cerebral causes.
The role of LRRK2 in the pathogenesis of PD has beenclarified, but its role in systemic inflammatory diseases isfar from clarification. It is well documented that LRRK2
is highly expressed in macrophages, monocytes, and neutrophils, strongly implicating its potential role as a regulator of immune responses [37]. Genomewide association
studies have identified LRRK2 as a risk factor for peripheral inflammatory responses in leprosy infection [38],bacterial infection and inflammatory bowel disease (IBD)[39]. The differences in the outcome of LRRK2-regulated
inflammation may be related to different types of LRRK2expressing immune cells and the signaling pathways
involved. LRRK2 has also been found to negatively regulate NFAT and reduce the susceptibility to DSS-induced
IBD, indicating that LRRK2 may inhibit the progressionof inflammation [29].
Currently, most studies of the role of LRRK2 in immunity and inflammation involve organs with abundant
expression of LRRK2, such as brain, kidneys, and lungs.
However, few have explored its role in the liver that has
a low LRRK2 expression. In inflammatory responses,
classic acute inflammatory cytokines are significantly
increased in the liver, brain, and peripheral circulation
when hepatic encephalopathy occurs. Studies have documented significant increases in IL-1β, IL-6 and TNF-α
in the brain and plasma in the TAA-induced rat ALF/
HE model [40] and marked elevation of pro-inflammatory cytokines such as TNF-α, IL-1 and IL-6 in HE
patients [41]. Likewise, the current study reported elevated levels of cytokines, such as IL-1β, IL-6, TNF- α, in
the TAA-induced ALF and HE mouse models (a classical type A HE model), which was positively correlatedwith the enhanced liver inflammatory response. Notably,
the Lrrk2−/−-HE mice reported the lowest survival rate,
highest blood ammonia level, and severest inflammatory cell infiltration in the hepatic portal area; no significant difference in survival rate was evident between
the Lrrk2G2019S-HE mice and WT-HE mice, suggestingthat LRRK2 plays a protective role in the TAA-induced
ALF and HE. However, it remains unraveled whether,Li et al. Journal of Neuroinflammation(2024) 21:123

despite its low baseline expression, LRRK2 enhances thehepatic inflammatory stimulation by regulating the activity of acute-phase inflammatory factors or otherwise.
The pathogenicity of LRRK2 is mostly conferred by thegain of kinase function [42], which can be enhanced by
G2019S mutation, the most common PD-related mutation [43]. In our study, the Lrrk2G2019S mice reported a
reduction in liver inflammation and mortality when compared with the Lrrk2−/− mice, and no significant protective effect when compared with the WT mice, which was
not reversed by G2019S mutation otherwise. A possible
explanation may be that the LRRK2 overexpression in the
Lrrk2G2019S mice cannot completely replace the normal
physiological functions of LRRK2.
Of interest, the current study documented different
histopathological changes in the liver and brain, which
may be attributed to the regional differences in LRRK2
expression in the TAA-HE mice. In the Lrrk2−/−-HE
mice, LRRK2 deficiency significantly attenuated the
TAA-induced injury to different brain regions, including
the PFC and the dorsal STR that regulate motor function, the ventral STR that involves self-reward, and the
HIP CA3 and dentate gyrus (DG) regions that modulate
learning and memory. The available literature evidencesthat LRRK2-mediated immunoreactivity can degrade themyelin sheaths [44] and that small myelinated nerve fibers are crucial for maintaining a balance between excitation and inhibition in the brain hemispheres [45]. In
the current study, the Lrrk2G2019S-HE mice reported asignificant degeneration of myelin sheaths in the STR,
especially the small myelinated nerve fibers, in which themice, under the stressful circumstances, displayed motor
coordination dysfunction in the challenging rotarod test.
The above results suggest that the difference in LRRK2
backgrounds in HE patients may indicate a clinical translational significance in elucidating the pathophysiologicalmechanisms of various symptoms.
Noteworthily, the current study also revealed a LRRK2
imbalance-induced synaptic structural impairment in the
TAA environment. In the available literature, controversies remain regarding the role of LRRK2 for synaptic vesicle transmission. Some previous studies have showed that
LRRK2 overexpression can decrease the release of neurotransmitters of dopaminergic neurons, which is partly
related to the LRRK2 overexpression-induced abnormal
protein deposition in the synaptic membrane and vesicle membrane structure, thus impairing vesicle transport[46]. Other studies, however, have found that LRRK2
knockout impinges clathrin-mediated neurotransmission and vesicle endocytosis [47]. In this study, the TAA
intervention induced the structural abnormalities in thevesicles of asymmetric and symmetric synapses in the
PFC. Of note, the Lrrk2−/− mice showed more typicalPage 16 of 19

vesicle structural abnormalities both at baseline and after
the TAA injection, especially in asymmetric synapses.
The results seem to indicate that the TAA-induced brain
damage may aggravate the injury to vesicle endocytosis
from LRRK2 knockout, resulting in synaptic transmission dysfunction, motor impairment, and lassitude.
Given the multiple functions of brain resident immune
cells, microglia form the frontline defense of the innate
immune system. Findings regarding the association
between microglia and LRRK2 remain controversial
however. Some studies have found that LRRK2 overexpression activates microglia [48, 49] while others suggestthat LRRK2 knockdown increases the microglial mobility, which may be attenuated by LRRK2 G2019S mutation
via the inhibition of focal adhesion kinase [50]. The current study found that LRRK2 knockdown failed to markedly activate the microglia and that inflammatory factors
increased in the brain of Lrrk2−/−-HE mice, which may
be attributed to the compensatory mechanisms of LRRK1[51]. In the Lrrk2G2019S mice, microglia were activatedbefore the TAA intervention and displayed an amoebic-like morphological change afterwards, indicating
functional depletion, which partially downregulates the
inflammatory factors. Therefore, despite the differences
in baseline expression in various tissues and organs,
LRRK2, under the external pathogenic stress, may exertdistinct or even opposite regulatory effects on the pathophysiological responses in systemic inflammatory diseases in order to protect the organs.
Autophagy, especially lysophagy, is most intensively
studied in LRRK2-regulated inflammation, but the roleof LRRK2 in different diseases remains controversial.
Some studies have reported that LRRK2 overexpression decreases autophagy in the BMDCs from Lrrk2
Tg mice that were exposed to an autophagy-inducing
microbe M. leprae [52] and that the most common
LRRK2 mutation, LRRK2-G2019S, significantly reduces
the transport of autophagosome in neurons in a kinasedependent manner [53], indicating that LRRK2 inhibits
autophagy. Other studies, however, argue that the lossof LRRK2 also leads to dysfunction of the autophagiclysosomal pathway [54]. Still others suggest that boththe enhancement and loss of LRRK2 expression canaffect the smooth progression of the whole process
of autophagy [55], indicating that the expression andactivity of LRRK2 must be appropriately moderated inorder to regulate the normal autophagy pathway. The
current study demonstrated an acute inflammatory
response in the liver and brain of the TAA-HE mice,
which was more serious in the former than in the latter,and an aggravated hepatic inflammation and autophagic
dysregulation by LRRK2 deletion, which significantly
increased the mortality of the Lrrk2−/− -HE mice. The

Li et al. Journal of Neuroinflammation(2024) 21:123

results of TEM reported more serious lysosomal disruption, autophagosomal accumulation, and significant
autolysosomal reduction in the Lrrk2−/−-HE mice thanin either the Lrrk2G2019S -HE mice or the WT -HE mice.
Western blotting revealed increased expression of classical protein markers of autophagy P62 and LC3B in
TAA-induced ALF and HE mice, suggesting impaired
autophagy flow [56]. Meanwhile, the expressions of
LAMP1, LAMP2 and pro-Cath-D increased but mature
Cath-D decreased in the ALF and HE mice, indicating aberrant functions of lysosomes and impaired
autophagic-lysosomal pathway. Collectively, the above
results suggest that the inhibition of autophagy flow
impairs its protection against acute inflammation in
the liver and that the knockout of LRRK2 can result ina severe blockage of the autophagic-lysosomal pathway.
Rab GTPases, as direct physiological substrates of
LRRK2, are required for regulating autophagy [16].
LRRK2 has been documented to interact directly withseveral Rab GTPases, such as Rab5, Rab7, Rab8, andRab10 [57]. In this study, we detected in lysosomes theexpressions of Rab5, Rab7, Rab8, Rab10, Rab29, andLRRK2, and the localization of Rab10 and LRRK2. The
results found no significant difference in the expressions of Rab5, Rab7, Rab8 and Rab29 among the groups
after the TAA intervention. However, the expression of
Rab10 increased and its phosphorylation decreased in
the Lrrk2−/−-HE mice, suggesting that in the TAA-HE
mice, LRRK2 may interfere with lysosomal homeostasis
by regulating Rab10 phosphorylation, thereby affecting
autophagy.
The results in this study reveal critical roles of LRRK2in TAA-induced liver inflammation and autophagy regulation. LRRK2 is known to regulate inflammatory progression through multiple signaling pathways, including
MAPK, NF-κB, and NFAT signaling pathways. In thepathogenesis of TAA-induced ALF/HE, the signaling
pathway for LRRK2-regulated inflammation and the roleof LRRK2 in the metabolism of blood ammonia remain
unilluminated and warrant further explorations. Currently, LRRK2-G2019S mutation is one of the most common mutations in PD and inhibitors targeting LRRK2
are in the clinical trials in the PD treatment [58]. However, whether LRRK2 inhibitors affect the normal physiology of peripheral tissues, especially the liver, awaits
further elucidation, despite that LRRK2 kinase inhibitors can effectively improve the clinical symptoms of PD
patients by inhibiting kinase activity. So far, many studies
have demonstrated that LRRK2 imbalance can seriously
impact the physiological functions of kidneys and lungs[59]. However, few reports are available to elucidate theeffect of LRRK2 on liver diseases, which is the original
intention of this study.

Page 17 of 19

In conclusion, to our knowledge, we demonstrate
for the first time that LRRK2 deficiency may exacerbate TAA-induced ALF and HE in mice, and explore
the involvement of LRRK2 in the inflammation andautophagic-lysosomal pathway in a TAA-induced HE
model. Given the important role of LRRK2 in regulating
TAA-induced liver and brain inflammatory responses inacute HE mice, sufficient care should be taken in evaluating the toxicities and indications of treatments targeting
LRRK2.

Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s12974-​024-​03125-4.
Supplementary Material 1.
Acknowledgements
The authors are grateful to all study participants and greatly appreciate
Professor Hongzhi Huang from School of Arts and Sciences of Fujian Medical
University, who carefully proofread and polished this manuscript.
Author contributions
XD. P. were responsible for conception and design; D. L, SF.Y., L. L and JR. G.
for the methodology; SF. Y., L. L., JR. G. and SM. H. for data acquisition andanalysis; D.L., SF.Y., L.L. and JR. G. wrote, reviewed, and/or revised the manu‑
script; D.L. and XD.P. supervised the study. Other authors are listed in order ofcontribution.
Funding
This work was supported by grants from National Natural Science Founda‑tion of China (No. 82071175); Natural Science Foundation (Key Project) of
Fujian Province, China (No. 2020J02022); Joint Funds for the innovation ofscience and Technology, Fujian Province (No. 2019Y9080, No. 2021Y9037,
No. 2021Y9064); Financial Special Project of Fujian Province (No. 2021XH009).
Availability of data and materials
Not applicable.

Declarations
Ethics approval and consent to participate
All animal procedures and experiments observed the National Guidance forAnimal Experiment and were approved by the Animal Ethics Committee of
Fujian Medical University (No. FJMU IACUC 2021-0313).
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Author details
1
Department of Neurology, Fujian Institute of Geriatrics, Center for Cogni‑
tive Neurology, Fujian Medical University Union Hospital, 29 Xinquan Road,
Fuzhou 350001, China. 2 Department of Gastroenterology, Fujian Medical
University Union Hospital, 29, Xinquan Road, Fujian 350001, China. 3 Fujian
Key Laboratory of Molecular Neurology, Fujian Medical University, 29 XinquanRoad, Fuzhou 350001, China. 4 Fujian Key Laboratory of Vascular Aging,
Fujian Medical University, Fuzhou 350001, Fujian, China. 5 Institute of Clini‑
cal Neurology, Fujian Medical University, 29 Xinquan Road, Fuzhou 350001,
China. 6 Fujian Clinical Research Center for Digestive System Tumors and Upper
Gastrointestinal Diseases, Fujian 350001, China. 7 Clinical Research Center
for Precision Diagnosis and Treatment of Neurological Diseases of Fujian
Province, Fuzhou 350001, China.

Li et al. Journal of Neuroinflammation(2024) 21:123

Page 18 of 19

Received: 14 August 2023 Accepted: 5 May 2024
22.

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