LL-37 Cathelicidin Antimicrobial Peptide – An Alzheimer’s Disease Therapy Candidate

Posted on October 30th, 2018

LL-37 or Human Cathelicidin Antimicrobial Peptide (CAMP) Background

The cathelicidin family of antimicrobial peptides are found in human and mammalian forms of life, as a mammal’s core tool to fight off microbial invasion of all kinds. The genes associated with the peptides have been thoroughly studied, are well-documented and have been compared between species. Other than a prehensile thumb, one of the most interesting unique aspects of humans is we only have one cathelicidin antimicrobial peptide gene: human gene LL-37.

LL-37 is expressed from epithelial cells, with current research identifying expression from the testis, skin, gastrointestinal system, respiratory system, and in immune cells like leukocytes (monocytes, neutrophils, T cells, natural killer NK cells, and B cells). LL-37 regulates inflammatory response and serves as a immune system “chemotaxi” – something like a taxi for immune cells – attracting immune system cells to the appropriate place where the cells are needed (like wound healing, infection sites, binding and neutralizing other antimicrobial peptides, etc).

MaxWell has the majority of our current intellectual property and R&D invested into this gene. It’s good for us to declare that we likely have some cognitive bias around this peptide. However, the fact is that LL-37 is the singular cathelicidin family peptide for humans. It is basic physics that so many disorders originate in function or disfunction of this single common pathway.

Cathelicidin LL-37 in the Context of Alzheimer’s Disease & Age Related ImmunoDementia (ARID)

Alzheimer’s Disease is a terminal diagnosis suffered by over 42 million patients globally – with no known cure. The handful of drugs currently prescribed to Alzheimer’s patients at best ameliorate symptoms (‘disease-modifying’ as opposed to ‘disease-curative’ treatments). Most ‘symptomatic therapies’ currently available are neurotransmitter-focused and fall into two classes: cholinesterase inhibitors, and the NMDA (glutamatergic) receptor agonist memantine. There are no FDA-approved drugs that address the underlying causes of Alzheimer’s disease, which remain poorly understood despite 110 years of research. [R1] Addressing the underlying cause of disease is our focus, here at MaxWell.

Alzheimer’s disease risk is known to correlate with the risks of several other chronic diseases, in particular metabolic syndrome / type 2 diabetes and cardiovascular disease; but these connections are understudied given the complexity of studying such associations. In practice, pharmaceutical drug development is done for only one chronic disease target at a time, although some FDA-approved drugs are repurposed for other conditions.

As of 2018, it has been 14 years since a new drug for Alzheimer’s disease was approved by FDA. The majority of the more than 400+ failed clinical trials pursued strategies to reduce the distinct, observable pathophysiological protein signatures of Alzheimer’s disease: (1) Aβ amyloid plaque accumulation in the brain, which is primarily extracellular (outside of neurons), and, much less commonly (2) phosphorylated tau protein aggregates, which form ‘neurofibrillary tangles’ within neuronal cells. Clinical trials aimed at reducing the burden of Aβ and tau aggregate protein deposits have so far failed to improve both patient cognition and function, as was required by the FDA for drug approval (until recently, when the FDA relaxed these criteria somewhat to focus therapies on functional endpoints).[R2]

Within the aging human brain, Alzheimer’s disease (AD) involves the assembly of β-amyloid (Aβ) peptides from soluble monomers into oligomers, fibrils and plaques [R3]. Studies of the spatiotemporal interplay between diffusible Aβ oligomers and fibrillar deposits as well as intracellular tau tangles have been aimed at investigating drivers of neuronal dysfunction, which are still not well understood. [R4, R5] The root physiological causes of sporadic (i.e., spontaneously arising) Alzheimer’s Disease—for which there is no known genetic predisposition, and which is the most common type of AD—remain unspecified, preventing the development of effective therapies to prevent, halt, or reverse the disease [R6].

Identification of physiologically relevant binding partners of Aβ that modulate fibril formation in vivo might yield new insights into causes of AD and help identify early instigators of Aβ accumulation and neurotoxic effects. A number of natural proteins that can bind to or interact with Aβ were identified by various assays [R7]; however, their roles in sporadic AD are often challenging to assess.

Literature reports exist, which in a connect-the-dots fashion, indirectly suggest that the biophysical activities and signaling functions of Aβ peptides and LL-37, the only cathelicidin-derived innate immune system peptide found in humans, are related in vivo. For instance, the vitamin D receptor (VDR) and retinoid X receptor (RXR) are both connected with AD; as well as with expression levels of LL-37. Vitamin D3 treatment was shown to reduce cerebral amyloid-β accumulation and improve cognition in a mouse model of AD, although the mechanism of action was not stated [R8], while RXR activation reduced neuronal loss and improved cognition in an aggressive mouse model of AD; but again, with no mention of cathelicidin involvement [R9]. It is never mentioned in the above AD papers that expression levels of the human CAMP gene that encodes hCAP-18, the protein precursor for LL-37 are upregulated by activation of VDR. [R10, R11] It is also never mentioned that RXR activation is obligate part of CAMP gene expression, although this has been shown. [R12, R13]

Another indirect connection between Alzheimer’s Disease mechanism and cathelicidin LL-37 is the hypothesis that sporadic AD is essentially ‘Type 3 Diabetes’ occurring in brain tissue [R14, R15]. In a 2015 study, it was shown that intraperitoneal administration of the murine cathelicidin peptide, CRAMP (which like LL-37 for humans, is unique in the mouse proteome) protects Non-Obese Diabetic (NOD) mice against the development of autoimmune diabetes. [R16] This treatment was motivated by the authors’ discovery that the genetic defect of NOD mice that creates susceptibility to autoimmune diabetes is a deficit in their ability to express cathelicidin; and this defect is more pronounced in female NOD mice, who have a higher incidence of disease as compared with males. In this animal model, immunomodulatory effects of the cathelicidin peptide, including effects on the phenotypes of white blood cells including macrophages, dendritic cells, T and B cells, reduced risks of inflammatory disease [R17]. What this shows, is that in vivo, the cathelicidin peptide is strongly immunomodulatory of white blood cell phenotypes: macrophages, dendritic cells, T cells, and B cells, were all switched from a proinflammatory, diabetogenic phenotype by the injection of cathelicidin peptide, to a noninflammatory, healing phenotype that prevented the development of autoimmune diabetes.

To date, most approaches to AD have relied on the supposition that pathological overexpression or hindered degradation of Aβ lays the primary foundation for disease. [R18]. Recently, it has been shown that the chronic underexpression of the cathelicidin LL-37, which normally opposes Aβ fibril formation, plays a key role in the pathological accumulation of Aβ, as will be discussed below [R19]. It is of course difficult for researchers to identify a systemic element that should be present, or perhaps should be better regulated, but is not; which is how we explain the fact that this central and important role of LL-37 in Alzheimer’s Disease was not discovered prior to 2017.

Recently, a multifaceted approach was taken to confirm and characterize, in vitro, the interactions between Aβ and LL-37, and inhibitory effects of LL-37 on Aβ oligomer/fibril formation. It was demonstrated that LL-37 and Aβ42 (residues 1-42 of the Aβ peptide), both individually toxic and proinflammatory to neuroblastoma cell line SH-SY5Y via the stimulation of microglial production of inflammatory cytokines, lose most of their cytotoxicity to neurons if the two peptides are co-incubated prior to being added to the cell culture media.

The interactions between LL-37 and Aβ peptides were investigated by SPR imaging (SPRi), a recent evolution of traditional SPR, which couples the label-free monitoring of molecular interactions by scanning angle SPR measurements with simultaneous CCD-based imaging of the whole surface for signal detection [R20]. The multi-array SPRi configuration improves the overall accuracy of the study by allowing simultaneous detection of signals originating from both positive and negative control ligands immobilized on the same chip. In addition, the real-time imaging of the entire SPR-biochip allows for verification of the quality and the optical properties of different ligands after their immobilization on the chip surface. To evaluate the aggregation state and the presence of soluble oligomers in the Aβ samples used for SPRi, capillary electrophoresis (CE) analysis was carried out. The inhibitory effect on fibril formation was demonstrated through transmission electron microscopy (TEM) by investigating fibril formation in quasi-physiological conditions. Conformational analyses of Aβ42 peptide in solution, in the absence and presence of LL-37, were carried out by circular dichroism (CD) spectroscopy.

In this work [R21], it was demonstrated that Aβ and LL-37 bind to each other specifically, and that LL-37 inhibits the adoption by Aβ of ordered β-type secondary structure. Additionally, a variety of different published findings were discussed that indicate a physiological interplay between, and potential co-regulation of these two peptides, as an aspect of human innate immunity that may affect the initiation and progression of Alzheimer’s Disease-related pathology.

Authors: Joshua McClure and Annelise Barron.

References

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