Molecular Psychiatry: The Kunming Institute of Zoology/Fudan team discovered for the first time that the antimicrobial peptide LL-37 accelerates the progression of Alzheimer's disease!

*For medical professionals only

In 1901, a German woman named Auguste came to the hospital to consult about symptoms of
memory loss.
Although Dr.
Alois, who saw her, was unable to provide a definitive cure, he kept a close eye on her condition
.
Until Auguste's death in 1906, Dr.
Alois dissected her brain and found strange plaques and fibrous tangles
in her brain.

Dr.
Alois, whose full name is Alois Alzheimer, was the first doctor
in the world to report Alzheimer's disease (AD).
The "strange plaques and fibrous tangles" in Auguste's brain are β amyloid (Aβ) and neurofibrillary tangles (NFT), based on the above two pathological phenomena, scholars have proposed the Aβ cascade theory and the abnormal phosphorylphosphochemical theory
of Tau protein.

But in fact, the pathogenesis hypothesis of Alzheimer's disease is much more than that, or is there a more upstream mechanism that leads to the formation of Aβ and NFT?

Recently, Lai Cheng and Hu Xintian from Kunming Institute of Zoology, Shu Yousheng of Fudan University, Michele Mazzanti of the University of Milan in Italy and others published important research results in the journal Molecular Psychiatry [1], they found that the human antimicrobial peptide LL-37 can activate chloride channel protein 1 (CLIC1), leading to glial overexcitation and neuroinflammation, and further induce NFT formation, Aβ42 elevation, Pathological changes in AD such as memory impairment.

These effects
can be suppressed when CLIC1 activation is blocked or when mice with knockout Clic1 gene are used.

This study reveals for the first time that LL-37 is an endogenous agonist of CLIC1, and proposes a new mechanism by which LL-37 can accelerate the progression of AD disease by activating CLIC1, providing new possibilities
for the development of AD-targeted drugs in the future.

Screenshot of the first page of the paper~

Amyloid (Aβ) has long been recognized by academia β as an important driver behind Alzheimer's disease (AD
).
However, reducing the Aβ burden may not imply a cure for AD or a slowdown in cognitive decline [2].

In the case of aducanumab, a drug that clears Aβ, two phase 3 clinical trials with the same design yielded completely opposite results: in the EMERGE study, the group of patients who received high-dose drug treatment had a 22% reduction in the Total Clinical Dementia Score Scale (CDR-SB) score, which means that the rate of worsening of disease symptoms slowed; In the ENGAGE study, CDR-SB scores increased by 2 percent in patients who also received high-dose drug therapy [3].

Perhaps we should think outside the box and re-examine Alzheimer's disease and focus on more possible AD pathogenesis and potential therapeutic targets
.

In recent years, there have been numerous articles on the association of microbial infections with AD, for example, periodontitis is associated with increased pro-inflammatory status and cognitive decline in AD patients [4], and repeated cycles activated by herpes simplex virus (HSV) can lead to inflammation of the brain and accumulation of neuronal and cognitive impairment [5].

Infection with these pathogens induces activation of resident immune cells (e.
g.
, microglia) in the brain, infiltration by peripheral immune cells, and neuroinflammation
.
According to the antimicrobial peptide (AMP) protection hypothesis, pathogen infection may also exacerbate Aβ deposition and AD progression [6].

Antimicrobial peptides are important effector molecules of the innate immune system, with broad-spectrum antibacterial activity, and have a strong killing effect on bacteria; In addition, some antimicrobial peptides have a killing effect on some viruses, fungi, protozoa and cancer cells, and can even improve immunity and accelerate the wound healing process
.
There are two main types of antimicrobial peptides produced in the human body: defensin family peptides and cathelicidin family peptides
.

In fact, Aβ itself is one of a series of antimicrobial peptides produced against pathogens.

That's right, the "monsters" that we want to defeat in every possible way are clearly "fighters" against the invasion of foreign enemies~

LL-37, the only member of the cathelicidin family of peptides, has been reported to be expressed at high levels in AD patients [7], which is undoubtedly imaginative: Will LL-37 also be like Aβ, obviously a good idea, but accelerate the progress of AD?

In addition, chloride channel protein 1 (CLIC1) levels have been found to increase by 60 percent in the hippocampus of patients with mild/moderate AD [8].

So what is the connection between LL-37 and CLIC1, and will they work together to advance AD?

First, it is necessary to determine whether the two interact directly, which the researchers confirmed through surface plasmon resonance (SPR) analysis
.
The equilibrium dissociation constant (KD) between LL-37 and CLIC1 is 5.
79x

10^-7M
。 In human microglia (HMC3) and human neuroblastoma cells (SH-SY5Y), western blot analysis (Western blot) showed that LL-37 increased CLIC1 expression and membrane translocation
in cells.

LL-37 can activate CLIC1 and promote chloride influx, and the higher the concentration, the stronger the effect, but the addition of IAA-94, an exogenous inhibitor of CLIC1, partially cancels the activation effect
of LL-37 on CLIC1.

After the addition of IAA-94 to 4 μM LL-37, the degree of chloride influx is between 2 μM LL-37 and 4 μM LL-37

At the same time, in HMC3, LL-37 activates CLIC1 to promote the production of various toxic factors, including IL-1β, IL-6, TNF-α and reactive oxygen species (ROS), while simultaneous administration of IAA-94 can significantly inhibit the pro-inflammatory effect
of LL-37.

IL-1β, IL-6, TNF-α and ROS levels were significantly reduced in the 4μM LL-37+IAA-94 group compared with the 4μM LL-37 group

All of the above undoubtedly proves that LL-37's "god operation" is based on activating CLIC1, and once the activation of CLIC1 is blocked or inhibited, LL-37 will be greatly reduced
.

The researchers wanted to further reveal the effects of LL-37 on the central nervous system of animals, such as whether LL-37 caused AD-like changes in mice.

The researchers injected 300 μg/kg of LL-37 into the hippocampus of wild-type (WT) and Chelic knockout (KO) mice, and performed the classic water maze test on the mice in which LL-37 administration significantly impaired learning and memory ability in wild-type (WT) mice
, although all mice completed their task after a 7-day training period.

The mice's time on stage reflects spatial learning and memory, while the time spent in the target quadrant reflects their long-term memory

Compared with the normal saline group, the administration of LL-37 in wild-type mice significantly upregulated the expression of ionized calcium binding adapter molecule 1 (IBA-1, marker of microglial activation) and glial fibrillary acidic protein (GFAP, marker of astrocyte activation), meaning that LL-37 induced an astrocyte/microglial activation-mediated inflammatory response
.

In addition, LL-37 induced an increase in the levels of inflammatory factors IL-1β, IL-6, and TNF-α in the hippocampus of wild-type mice, the death of neurons in the dentate gyrus of the hippocampus, a decrease in the number of dendritic spines in the hippocampus, and a decrease in
synaptophysin expression.
The response to LL-37 induction in knockout mice was greatly reduced, and it was clear that LL-37 accelerated AD progression in mice by its specific interaction
with CLIC1.

So will LL-37 have an impact on a close human relative, the monkey? The researchers injected LL-37 into the ventricles of three rhesus macaques, and after 6 months, the monkeys were sacrificed for AD pathology studies
.

Hematoxylin-eosin (HE) staining showed that LL-37 caused severe brain tissue damage in monkeys, including loose brain tissue structure and neuronal
disorganization.
Magnetic resonance imaging (MRI) showed marked dilation of the lateral ventricles, one of the important anatomical changes in AD, indicating significant atrophy of brain tissue after LL-37 administration.

From left to right: coronal, sagittal, cross-sectional; The red arrow points to the lateral ventricle

At the same time, immunochemical analysis showed that LL-37 induced the formation of neurofibril tangles (NFT) in the prefrontal cortex (PFC), entorhinal cortex (EC) and hippocampus, as well as increased
expression of amyloid-42 (Aβ42) β.
NFT, formed by aggregation of the hyperphosphorylated microtubule-associated protein Tau, is highly neurotoxic and is a core pathological hallmark of AD [9].

Previous studies have shown that NFT load in the brains of AD patients is positively correlated with cognitive impairment [10].

Similar to mouse results, LL-37 induced activation of microglia and astrocytes in the monkey frontal cortex, entorhinal cortex and hippocampus, reduced the number of neurons, and increased IL-1β, IL-6, and TNF-α levels
in the hippocampus.

It has been previously reported that synaptic loss in AD is highly correlated with the degree of dementia [11].

So what about synapses in monkeys injected with LL-37? Western blot analysis showed that LL-37 significantly reduced hippocampal synaptosin expression, while CLIC1 expression was significantly upregulated
.
Golgi staining showed a decrease in dendritic spines in
the hippocampus.
In addition, TUNEL staining showed that LL-37 caused apoptosis
in monkey brain tissue cells.

SYP: Synaptophysin
.
β-Actin: Relatively constant expression in tissues and cells, often used as an internal reference when detecting changes in protein expression levels

Based on the above findings, the researchers proposed a new pathogenesis
of AD.
Typically, human antimicrobial peptide LL-37 expression levels are low, often at nM levels, and upregulated to 10 μM
when infection and inflammation occur.
LL-37 induces glial cell overexcitation, neuroinflammation and toxicity by promoting CLIC1 expression, membrane translocation and activation, thereby causing neuronal death and promoting the progression of
AD.

Perhaps in the near future, blocking the interaction between LL-37 and CLIC1 could bring new hope
for AD treatment.
Let's look forward to it together~

References:

[1] Chen X, Deng S, Wang W, et al.
Human antimicrobial peptide LL-37 contributes to Alzheimer's disease progression [published online ahead of print, 2022 Sep 22].
Mol Psychiatry.
2022; 10.
1038/s41380-022-01790-6.
doi:10.
1038/s41380-022-01790-6.

[2] Gosztyla ML, Brothers HM, Robinson SR.
Alzheimer's Amyloid-β is an Antimicrobial Peptide: A Review of the Evidence.
J Alzheimers Dis.
2018; 62(4):1495-1506.
doi:10.
3233/JAD-171133.

[3]  style="white-space: normal;margin: 0px;padding: 0px;box-sizing: border-box;">[4] Ide M, Harris M, Stevens A, et al.
Periodontitis and Cognitive Decline in Alzheimer's Disease.
PLoS One.
2016; 11(3):e0151081.
Published 2016 Mar 10.
doi:10.
1371/journal.
pone.
0151081.

[5] Cairns DM, Itzhaki RF, Kaplan DL.
Potential Involvement of Varicella Zoster Virus in Alzheimer's Disease via Reactivation of Quiescent Herpes Simplex Virus Type 1.
J Alzheimers Dis.
2022; 88(3):1189-1200.
doi:10.
3233/JAD-220287.

[6] Moir RD, Lathe R, Tanzi RE.
The antimicrobial protection hypothesis of Alzheimer's disease.
Alzheimers Dement.
2018; 14(12):1602-1614.
doi:10.
1016/j.
jalz.
2018.
06.
3040.

[7] Lee M, Shi X, Barron AE, McGeer E, McGeer PL.
Human antimicrobial peptide LL-37 induces glial-mediated neuroinflammation.
Biochem Pharmacol.
2015; 94(2):130-141.
doi:10.
1016/j.
bcp.
2015.
02.
003.

[8] Averaimo S, Milton RH, Duchen MR, Mazzanti M.
Chloride intracellular channel 1 (CLIC1): Sensor and effector during oxidative stress.
FEBS Lett.
2010; 584(10):2076-2084.
doi:10.
1016/j.
febslet.
2010.
02.
073.

[9] Ballatore C, Lee VM, Trojanowski JQ.
Tau-mediated neurodegeneration in Alzheimer's disease and related disorders.
Nat Rev Neurosci.
2007; 8(9):663-672.
doi:10.
1038/nrn2194.

[10] Arendt T, Stieler JT, Holzer M.
Tau and tauopathies.
Brain Res Bull.
2016; 126(Pt 3):238-292.
doi:10.
1016/j.
brainresbull.
2016.
08.
018.

[11] Clare R, King VG, Wirenfeldt M, Vinters HV.
Synapse loss in dementias.
J Neurosci Res.
2010; 88(10):2083-2090.
doi:10.
1002/jnr.
22392.
ADtaxi

The author of this article Davina

Responsible editorDai Siyu