miRNA in Alzheimer's Disease

Alzheimer’s Disease (AD) is a neurodegenerative disease that is projected to affect more than 1.3 million people in Canada in the near future [1]. It is characterized by increased inflammation, oxidative stress and a loss of neurons in several brain regions including the hippocampus and the prefrontal cortex gradually leading to memory loss and cognitive impairment. Some of the main changes in the proteome leading to Alzheimer’s Disease include hyperphosphorylation of the microtubule-associated tau proteins and overexpression of β-amyloid plaques due to a dysregulation in the splicing of its precursor protein, the amyloid precursor protein (APP) [2]. AD detection is currently limited to imaging techniques such as fMRI or PET scans [3] during the late stages of the disease so preventive measures are then difficult to implement and no treatments are available. miRNAs, small non-coding RNAs that regulate gene expression have recently been implicated in Alzheimer’s Disease in that their dysregulation leads to the proteomic changes that result in AD. They play a role in the disease onset and their abundance in body fluids such as the blood and the cerebrospinal fluid (CSF) make them potential biomarkers for early detection of the disease using everyday laboratory techniques such as northern blot and RT-PCR [4]. miRNAs act through the RNA interference pathway which can be used to silence gene expression. This technique can be used as a potential treatment for AD by targeting the aberrantly expressed proteins to alleviate some symptoms of the disease during its early phases [5].

I- Differential expression of miRNA in the AD brain

1- miRNA mechanism and general function

miRNAs are small non-coding RNA sequences. Via a process called RNA interference, miRNAs are responsible for post-transcriptionally silencing gene expression by base pairing to complementary stretches of mRNA and recruiting the miRISC complex (miRNA-induced silencing complex) which prevents translation or initiates mRNA degradation depending on the degree of complementarity between the mRNA and its specific miRNA strand [6]. miRNA-mediated gene regulation affects cellular function via the resulting differential protein expression. Such functions include proliferation, apoptosis, development as well as regulation of the cell cycle [7]. Neural miRNAs such as miR-106 have been shown to affect synaptic development, and in consequence, development of the central nervous system [8] and dysregulation of miRNAs would lead to severe defects such as cognitive impairment in the case of miR-106.

APP Processing into β-Amyloid in Alzheimer’s Disease
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taken from Schonrock, N. et al., MicroRNA networks surrounding APP and amyloid-β metabolism
- Implications for Alzheimer’s Disease. Experimental Neurology 235:447-454 (2012)

2- Aberrantly expressed proteins in AD

Alzheimer’s Disease can have familial or sporadic origins, the latter being the most common. AD, then, is rarely due to hereditary genetic defects. the “amyloid cascade hypothesis” is the dominant hypothesis in AD and it is characterized by an accumulation of β-amyloid plaques as well as the presence of neurofibrillary tangles [9].

The amyloid precursor proteins (APP) have different isoforms and they can be processed via different proteins. One such protein, BACE1, cleaves APP into β-amyloid plaques whose aggregation can result in negative effects such as axon pruning and neuronal cell death [10].

Tau proteins play a role in stabilizing axonal microtubules. In the AD brain, these proteins are hyperphosphorylated due to an overexpression of tau kinases such as ERK12 which allows them to dissociate from microtubules, destabilizing them in the process; they then aggregate to form neurofibrillary tangles ultimately leading to cell death [11]. Other proteins such as pro-inflammatory cytokines are overexpressed, leading to the activation of the inflammatory cascade that can result in neurodegeneration [2].

miRNAs are abundantly expressed in the brain and they mediate important neurobiological functions that can lead to neurodegenerative diseases when they are dysregulated. Indeed, in the aging brain, miRNAs, prone to having a differential expression pattern [12], could be a risk for AD onset [2]. Recent research has started to implicate miRNAs with some of the most important proteins in Alzheimer’s Disease pathology.

3- Pathways affected by miRNA dysregulation

Alzheimer’s disease mostly affects people over the age of 65. Aging alters miRNA levels in the brain due to oxidative stress [13], which is an important mechanism in neurodegenerative diseases that causes an imbalance between production of reactive oxygen species (ROS) and the biological system’s ability to detoxify these reactive intermediates using antioxidant systems [14]. The Differentially expressed miRNAs affect multiple

Different Pathways Affected by Aberrant miRNA Expression
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taken from Tan, L. et al., Non-coding RNAs in Alzheimer’s disease. Molecular Neurobiology 47:382-393 (2013)

pathways which eventually lead to an AD brain such as the amyloidogenic, tau toxicity and inflammatory pathways. RT-PCR experiments on transgenic mouse models for AD (SAMP8) have shown that out of the all the miRNAs in the brain a few were upregulated or downregulated compared to control mice populations [15].

In the amyloidogenic pathway, β-amyloid overexpression which leads to β-amyloid plaques can be due to an increase in BACE1 to process more APP or a defect in β-amyloid degradation. BACE1 has target sites for several miRNAs including miR-107 miR-195. Their decrease in AD correlates with an increase in BACE1 which leads to more APP cleavage and consequently more β-amyloid proteins. these miRNAs are then regulators of BACE1 activity [16]. Similarly, it has been shown that miR-16 directly target APP to inhibit them. Experiments using miR-16 mimics in SAMP8 mice show a significant decrease in APP levels when injected. Coincidently, miR-16 is downregulated in AD while APP is overexpressed causing cognitive impairment and β-amyloid plaque deposition [9].

As for the tau toxicity pathway, BAG2, a chaperone that mediates the degradation of hyperphosphorylated tau is usually regulated via miR-128 [17]. In AD, this specific miRNA is downregulated, leading to an accumulation of hyperphosphorylated tau. Recent data has shown that miR-132, an miRNA that mediates alternative splicing of tau is downregulated in neuropathological conditions allowing differential splicing, a common phenomenon in AD, to occur [18].

The nuclear factor kappa B (NF-κB) is a transcription factor that mediates the production of cytokines. This Inflammatory signaling pathway is essential for repair of neural mechanisms. Incidentally, AD is associated with an overexpression of NF-κB-activated miRNAs. These miRNAs in turn inhibit certain proteins such as complement factor H (CFH) which is an immune system repressor. The complement pathway is a part of the immune system that stimulates inflammation [2]. CFH is an important regulator of complement. Post-mortem studies AD brains have shown a downregulation of CFH correlated to an overexpression of NF-κB-activated miRNAs such as miR-125 and miR-146a [19], ultimately leading to neurodegeneration through an increase in inflammation. The inflammatory pathway has been linked to many neural diseases such as Autism (see Immune Responses in Autism).

II- miRNAs as biomarkers for early detection

1- presence of miRNAs in the blood circulation and the CSF

Currently, imaging techniques performed on late-stage AD patients are one of the few usable diagnostic techniques in Alzheimer’s Disease. This is mainly due to our inability to identify AD biomarker proteins such as β-amyloid or hyperphosphorylated tau through non-invasive techniques.

miRNAs provide a decent alternative as they are usually highly abundant in the blood and the CSF and recent data has demonstrated that miRNA levels in these fluids fluctuate similarly as those studied in brains of AD patients [20]. Notably, miRNAs are already being used to as biomarkers in the blood to diagnose different diseases such a cancer or exposure to certain nanoparticles [21]. Several papers have shown that miR-146a, part of the inflammatory pathway in AD, is present and upregulated in both the brain and monocytes [22] [23]. AD-specific miRNAs in the blood and the CSF can then be used for AD detection.

One limitation is whether the miRNAs in the blood appropriately reflect those in the AD brain despite similar responses in different pathologies.

2- miRNA for early detection of AD

Potential Advantages of miRNA-mediated Early Detection
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taken from Kato, M. et al., MicroRNA Circuits in Transforming Growth Factor-β Actions and Diabetic Nephropathy.
Seminar in Nephrology 32:253-260 (2012)

miRNAs have certain advantages over other potential biomarkers for neurodegenerative diseases. They are relatively stable and highly specific for diseases such as AD. miRNAs participate early on in the post-transcriptional stages of gene expression. Changes in miRNA levels would precede any effects they might cause, making them perfect candidates for detecting diseases early on during their asymptomatic stage. Early detection allows for health-care providers to intervene during a stage where the best treatments and chances of modifying the disease could be provided [24].

One paper by Zhang et al (2013), proposes a mechanism linking the onset of cognitive impairment, a condition often linked to AD, to a dysregulation in miR-106a. The JAK2/STAT3 pathway is a transcription activator that plays an important role in in the development of the central nervous system. It has often been linked to memory impairment and AD as its inhibition can be achieved through either age-dependent factors or high levels of β-Amyloid in hippocampal neurons, one the main brain areas for memory formation.

The authors used ovariectomized (OVX) mice to simulate post-menopausal hormonal changes in humans and found out that normal mice performed better than OVX mice in tasks such as the Morris water maze. miR-106a appears to play an important role in regulating the JAK2/STAT3 pathway. Western blot and PCR experiments showed abnormal levels of both miR-106a and STAT3 in the older OVX mice. Luciferase reporter assays confirmed an interaction between miR-106a and STAT3 as overexpressing this miRNA, as is the case in AD, seemed to downregulate the activity of the JAK/STAT pathway.

This paper is particularly important as it briefly hints about the potential for using specific miRNAs as biomarkers for early detection of AD even though it doesn’t explore the matter in details. Detecting high levels of miR-106a in hippocampal neurons can help healthcare providers diagnose patients with cognitive impairment which can be useful as a potential screening test for AD. This technique seems to be highly specific for hippocampal neurons. miRNAs’ main advantage as biomarkers are the non-invasive techniques used for diagnosis and this research fails to mention whether miR-106a levels in easily testable body fluids such as blood fluctuate similarly to those in hippocampal neurons in the case of old OVX mice [8].

3- miRNA detection techniques

As mentioned previously, miRNAs are highly abundant in body fluids that are routinely investigated using common laboratory tests. Recent scientific advancement allowed for the identification of miRNAs using techniques such as Real-time PCR, northern blotting or microarrays. These techniques allow for a quick and non-invasive diagnosis of AD. Recent studies used screening tests to detect miRNAs related to mild cognitive impairment, a disease frequently associated with AD and using longitudinal studies the results were shown to be mostly accurate in predicting neurodegenerative diseases as well as detecting age-related brain changes [25]. Some of the limitations of these techniques cause sensitivity problems due to the short size of miRNAs [24].

III- miRNA-based treatments

1- Targeting of the aberrant Proteins via miRNAs

miR-16 Mimics Reduce Expression of APP Proteins
Image Unavailable
taken from Liu, W. et al. MicroRNA-16 targets amyloid precursor protein to potentially modulate
Alzheimer’s-associated pathogenesis in SAMP8 mice. Neurobiology of Aging 33:522-534 (2012)

The RNA interference pathway is commonly used to silence specific proteins by targeting them with miRNAs mimics that usually regulate these proteins in vivo. Several proteins in Alzheimer’s Disease are overexpressed due to a downregulation of specific miRNAs. Multiple studies have tried overexpressing these miRNAs and have observed a downregulation of the proteins in question. Notably, miR-195 can target BACE1 to downregulate β-amyloid proteins. In AD, miR-195 is usually downregulated and it’s overexpression can potentially be used a therapeutic strategy [16]. This technique can be applied to any downregulated miRNA previously seen such as the miR-16 which usually downregulates APP.

One of the major limitations of RNAi therapeutic approaches remains the delivery method in vivo, with intranasal delivery being the most plausible and if it is not specific enough it can potentially interfere with the endogenous RNA interference pathway [2].

2- Antagomirs against the overexpressed miRNAs in AD

Antagomirs or anti-miRs are small synthetic RNAs used to prevent miRNA binding to their target. They can be used as a potential therapeutic approach for Alzheimer’s Disease by trying to regulate the aberrantly expressed miRNAs that lead to overexpressed proteins.

A recent paper showed an increase in the levels of BDNF, a protein found in low levels in AD patients involved in synaptic plasticity and memory by targeting miR-206, an miRNA that regulates BDNF. More specifically, this miRNA, overexpressed in AD was inhibited by AM206, an antagomir. The increase in BDNF was accompanied by synaptogenesis and neurogenesis. Antagomirs can then be used to alleviate some of the symptoms of AD assuming the disease was detected early on [26].

Related Pages

Alzheimer’s Disease and Stroke
Alzheimer's Disease Models
Immunology of Alzheimer's Disease
Sex and Gender in Alzeheimer's Disease
Shock Therapy Treatment in Alzheimer's Disease
Genetics Behind Alzheimer's Disease
Effects of Lifestyle on the Prevalence of Alzheimer's Disease

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