26 October 2020
We looked forward to Lowry’s “Alzheimer's disease: protective effects of Mycobacterium vaccae, a soil-derived mycobacterium with anti-inflammatory and anti-tubercular properties, on the proteomic profiles of plasma and cerebrospinal fluid in rats” publication in the Journal of Alzheimer’s Disease  as an often-overlooked possibility towards Alzheimer’s disease (AD) genesis.
The thought that AD might be of a mycobacterial etiology has recently gained traction, but has been too little expressed. Mawanda and Wallace simply cite that “amyloidopathy—a condition characterized by elevated levels of serum amyloid and by amyloid deposition and aggregation in tissues—is a frequent occurrence in several acute and chronic systemic inflammatory conditions, especially chronic infections like tuberculosis.” . Several studies have already shown a “neuroprotective affect” using anti-mycobacterial antibiotics [3,4], and evidence suggests that patients aged 50-64 with infection with M. tuberculosis have a significantly higher risk of dementia .
And now still other evidence presents itself.
The discovery that the apolipoprotein (APOE) ε4 allele happens to be the strongest genetic risk for the most common form of AD led in turn to the consideration that the identification of the noncoding or microRNAs that control APOE as well as brain lipid metabolism might provide both a way to identify AD diagnostically and a way to treat it. But true understanding of genetic test results also requires attention to potential detail to avoid inaccurate results by exclusion. For example, APOE ε4 alleles themselves are known to show a distinct increase in tuberculosis (TB) . And should this finding carry through to the various microRNAs established to be important to AD, still another argument can be made for the mycobacterial origins of AD.
The discovery of the first microRNA (miRNA) over 20 years ago has ushered in a new era in molecular biology. miRNAs are attractive molecules to be considered as one of the blood-based biomarkers for neurodegenerative disorders such as AD. And searching for non-invasive AD biomarkers is currently one of the most rapidly growing areas in AD research . miRNA profiles have been extensively studied as potential bio-indicators for AD in blood, cerebrospinal fluid (CSF), and brain tissue. However, due to the high variability between the reported data, stemming from the lack of methodological standardization and the heterogeneity of AD, narrowing the field of the many miRNA to just a few of the most promising biomarker candidates has been slow to realize. Of special interest in this respect were two recent studies: first a large, 2019 review by Nagaraj et al. which found only 3 miRNAs: miRNA-146a, miRNA-125b, and miRNA-135a that were consistently reported in AD blood, CSF, and brain tissue . This was after doing an extensive literature review that showed that out of 137 miRNAs found to be altered in AD blood, 36 have been replicated in at least one independent study, and out of 166 miRNAs reported as differential in AD CSF, 13 have been repeatedly found. But only 3 miRNAs were consistently reported as altered in three analyzed specimens: blood, CSF, and the brain (hsa-miRNA-146a, hsa-miRNA-125b, hsa-miRNA-135a).
Also, just prior to the Nagaraj paper, Yang’s study, which also found miRNA-135, in addition to miRNA-193b and miRNA-384, as potential early biomarkers for the early diagnosis of AD .
What will become obvious here, is that all of the proposed miRNA markers from both the Nagaraj and Yang studies find their miRNA counterparts in similar biomarker diagnostics for TB and the mycobacteria.
The increase in miRNA-384, noted by Yang to be indicative of AD, is also significantly increased in mice with experimental autoimmune encephalomyelitis (EAE) using complete Freund’s adjuvant containing heat-killed Mycobacterium tuberculosis H37Ra. The use of heat-killed TB is mandatory in the induction protocols of many experimental models of EAE .
Both Nagaraj’s and Yang’s studies point to the importance of miRNA-135a in AD diagnosis with Yang pointing to the fact that in his study serum levels of miRNA-135a, increased significantly like miRNA-384 in patients with mild cognitive impairment and AD. Also, it was miRNA-135 that yielded the highest diagnostic accuracy in discriminating mild cognitive impairment patients from normal controls. All of Yang’s preferred AD markers in the serum (miRNA-135, miRNA-193b, and miRNA-384) were found previously to regulate the expression of amyloid-β protein precursor (AβPP) which eventually becomes amyloid-β (Aβ), whose amyloid fibrillar form is the primary component of amyloid plaques found in the brains of AD patients.
Yang proved miRNA-135a to be abnormally expressed in AD , but its value toward detecting tuberculous meningitis (TBM) had to wait for Lin et al.  who both confirmed its miRNA-135a’s diagnostic value for TBM as well as its worth in indicating prognosis. To Lin, miRNA-135a exerts a vital effect on the development, progression, as well as prog¬nosis of TBM. In fact, miRNA-135a exerts a vital effect on the development, progression, as well as prog¬nosis of TBM .
TBM remains a serious, often undetected health threat. Chronic neuropsychological sequelae may occur even after appropriate treatment, often in the form of cognitive impairment as seen in AD , though the mechanisms that lead to and mediate cognitive and behavioral outcomes in TBM remain unclear . Due to its hidden onset and long course of disease, clinical manifestations and laboratory tests lack specificity . It is because the diagnosis of TBM mainly rests on pathogen detection, which has the limitations of long cycle, poor sensitivity, and low specificity that delay in diagnosis and treatment result in the high fatality and high morbidity of TBM . Therefore, it is of great significance to explore methods for the early diagnosis of TBM . One of these is through the diagnostic value of miRNA-135a for TBM and its association with prognosis.
While Yang struggled to explain why AD biomarker miRNA-193 decreased significantly in the serum of those with mild cognitive impairment and AD, Lyu posited that such a dip in miRNA-193 could be expected as a manifestation of the standard differentially expression of miRNA-193 were such mental impairments due to active infection with Mycobacterium tuberculosis (Mtb) , a disease which Lyu validated miRNA-193 to be specifically expressed in.
As for Nagaraj et al.  who found only 3 miRNAs: miRNA-146a, miRNA-125b, and miRNA-135a that were consistently reported in AD blood, CSF, and brain tissue these also find their counterparts in other studies.
microRNA-146a and microRNA-193
Besides being premier AD marker candidates, Miotto et al. found both Nagaraj’s miRNA-146 and Yang’s miRNA-193 to be among the 15 miRNAs identified as a signature for discriminating between healthy controls and patients with pulmonary TB .
Despite the existence of various anti-mycobacterial therapies, TB remains one of the world’s major causes of illness and death. Approximately one third of the world’s population is thought to be infected with Mtb, and more than 9 million develop “active” TB each year . As the first line of host defense, macrophages are responsible for intracellular killing of Mtb. Paradoxically, they are also its principal target cells. It has been well-accepted that Mtb has evolved a serious of strategies to subtly modulate host immunity and create a microenvironment favoring its replication and growth—of which, regulation of miRNAs expression is considered as an important one. miRNAs are non-coding, single-stranded RNAs of ∼22 nt in length that regulate gene expression. In mycobacteria-infected macrophages, miRNA-146a is not only robustly increased, but leads to a higher mycobacterial burden in infected macrophages. miRNA-146 expression significantly increased in mouse macrophages post Mycobacterium bovis Bacille Calmette-Guérin infection (unpublished data), suggesting that miRNA-146a may play a role in TB-associated inflammation .
Besides being proposed as a test to differentiate between healthy individuals and those with pulmonary TB, miRNA-146a has been proposed as both a biomarker for human and cattle TB . miRNA-146a can increase up to 20-fold in mycobacterial infection, which promotes mycobacterial survival in macrophages by suppressing nitric oxide production .
TB is an inducer of miRNA-125b . And Fu et al. found miRNA-125b increased in the serum of TB patients .
Similarly, lipomannan from virulent Mtb stimulated a high expression of miRNA-125b in human macrophages . Lipomannan is a glycolipid in TB’s cell wall which plays a critical role in the pathogenesis of TB .
The etiology of AD is still of course unknown, but it is good to see the substantial progress through different avenues such as the paper by Lowry and colleagues  to better define it.
Lawrence Broxmeyer, MD
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