Sporadic AD is a complex disease with many risk factors, including genetic, environmental, and lifestyle factors (higher education, reading, taking care of family, smoking, etc.) (Bhardwaj et al. 2017). Whether mitochondrial polymorphisms are also risk factors is largely unknown. Recent studies suggest that mitochondrial mutations and other disturbances have an important effect on the pathogenesis of AD. Many mitochondrial haplogroups have been linked to AD as factors capable of conferring increased or decreased risk of AD. Given the role of mitochondria in cell death, their implication in neurodegenerative disorders, including AD, seems plausible. Multiple studies have been conducted to inspect the potential causative relation between mtDNA and AD. In this study, we evaluated the possible association between AD and mitochondrial haplogroups or polymorphisms in the Tunisian population by genotyping mitochondrial polymorphisms dispersed across the whole mitochondrial genome, using a cohort of 58 AD patients and 196 controls comparable in gender, age, and ethnicity.
To our knowledge, this investigation is the first case-control study related to AD and mtDNA in Tunisia, home to an admixed population from North Africa. The results showed that none of the observed mitochondrial haplogroups in our population had a significant association with AD. However, we observed that two mitochondrial variants, A5656G and A13759G, were statistically associated with AD.
In this study, we explored Tunisian mtDNA in relation to AD. The results showed a large variety of haplogroups, creating a mosaic structure composed of 41 haplogroups in our cohort (254 individuals only) and representing a high level of genetic diversity in the Tunisian population. This genetic diversity in Tunisia can be explained by a heterogeneous gene pool caused by invasions from different populations: Phoenicians, Byzantines, Arabs, Romans, Ottomans, Vandals, Andalusians, etc. Most of these populations have left a genetic imprint on the Tunisian population. In addition, the arrival of thousands of Europeans (from France, Italy, Spain, Austria, Belgium, Greece, Russia, and Malta) in Tunisia during the colonisation (about 1881–1921) further enriched the genetic pool (Cherni et al. 2016). As has been noted in previous studies, the long and rich history of Tunisia created a specific genetic pool. This genetic heterogeneity was observed both in different Tunisian regions and in populations of the same ethnic background. In our study, we found the same haplogroups as previously described in other studies (Elkamel et al. 2018; Costa et al. 2009). The major haplogroups found belonged to the West Eurasian lineages (H, T, U, K, N, V, I, W, J, X), with a predominance of the sub-haplogroup H2a. In general, haplogroup H is the most frequent European lineage in North Africa. The highest frequencies were found in Berber- and Arab-speaking Moroccans (37% and 34%, respectively) (Rando et al. 1998). Approximately the same percentage was found in our study, as 35.4% of our cohort had haplogroup H. Some previous studies have reported an association between haplogroup H and AD. For example, in Asturias (northern Spain), a significant association was found between haplogroup H and sporadic AD (Coto et al. 2011). In Italy, a specific sub-haplogroup of H (H5) was identified as a risk factor for AD (Santoro et al. 2010b). Despite the predominance of sub-haplogroup H2a in our population, we did not find any significant value for an association with AD.
The Eurasian lineage U was also present in our population (10.6%). This mitochondrial haplogroup has a large distribution in Europe and southwest Asia (Sahakyan et al. 2017). In Caucasian Americans, haplogroup U (p = 0.007) showed a differential effect in AD patients according to gender; it had a protective effect in females, and it increased the risk of developing AD in males (van der Walt et al. 2004). The North African-specific lineage U6 was present (5.5%) and constituted, together with the North African haplogroup M1, 6.7% of our population.
In agreement with Frigi et al. (2017), who described the Tunisian genetic pool by studying 15 Tunisian population groups distributed across the country, our cohort contained two North African haplogroups, M1 and U6, with a percentage of 7.1%. For the sub-Saharan lineages (haplogroup L), we found almost the same percentage (22.8%) as Frigi et al. (2017) (23%). Sub-Saharan haplogroup L1 showed an increased risk of developing dementia in African Americans with lower plasma levels of Aβ (Tranah et al. 2014).
Mitochondrial haplogroup T was among the Eurasian lineage in our cohort (9.8%) and was presented by both sub-haplogroups T1 and T2. In conflicting reports, mitochondrial haplogroups U and T have been implicated as conferring both a decreased and an increased risk of developing AD (Ridge and Kauwe 2018). In our case, the results showed no huge difference between patients and controls regarding haplogroup T (T1 +T2), which was present in 10.3% of patients and 9.6% of controls.
In many studies, evidence was found for an association between some specific haplogroups and AD, either as a risk factor or as a protective one. In our cohort, we have a mix composed of North African, sub-Saharan, and Eurasian lineages. This mixed population facilitates the evaluation of mitochondrial haplogroups in relation to AD. In our cohort, we have almost all mitochondrial haplogroups that were indicated in previous studies as being associated with AD. However, in our case, none of the haplogroups had a significant association with AD. These findings support the idea that haplogroup implication in AD is not strong enough to explain the risk of AD. Our study is in agreement with previous studies showing no definitive association of AD with a specific mitochondrial haplogroup (van der Walt et al. 2005; Elson et al. 2006; Mancuso et al. 2007; Mancuso et al. 2009; Fachal et al. 2015; Ridge and Kauwe 2018; Monzio Compagnoni et al. 2020; Zhunina et al. 2020). In fact, investigating the implication of mitochondrial haplogroups in AD pathology is an alternative way of determining the involvement of the mitochondrial genome. It is an approach to analysing the frequencies of mitochondrial polymorphisms that could affect AD pathology by altering mitochondrial respiration and causing overproduction of reactive oxygen species (ROS). This means that a group of individuals or a population with the same mitochondrial genotype has the same predisposition to apoptotic processes (Mancuso, Orsucci, et al. 2008). The analysis of the distribution of haplogroups in controls and AD patients is a way to determine whether a particular mitochondrial haplogroup is associated with the risk of AD by being more vulnerable to oxidative stress than other mitochondrial haplogroups.
However, the real involvement of mtDNA and its haplogroups in AD is still under discussion. Two theories are possible. The first one is that oxidative stress and mitochondrial dysfunction are responsible for neurodegenerative pathologies and have a primary role in AD. The second theory is that neurodegenerative pathologies are the consequence of cell death and that the decline in mitochondrial function is caused by other factors (Mancuso et al. 2006). To our knowledge, no previous findings, except one study (Coto et al. 2011), were replicated to date.
Classically, it has been difficult to study the direct implication of mtDNA mutations in AD patients, and many studies have produced non-conclusive results (Elson et al. 2006). Therefore, genetic investigations started to focus on single polymorphisms rather than haplogroups (Mancuso et al. 2009). Since some mitochondrial mutations were identified in association with AD (Shoffner et al. 1993), we changed the strategy from haplogroups to mitochondrial SNPs. Our findings showed two large effects: A5656G (p = 0.03821, OR = 10.46) and A13759G (p = 0.03719, OR = 10.78); both SNPs were not mentioned before as a risk factor for AD.
According to the human phylogenetic tree, A13759G is located on the branch point of two sub-haplogroups, H11 and j2a2. A13759G is located in subunit 5 of the NADH dehydrogenase locus. This enzyme belongs to complex I (NADH dehydrogenase, NDU) of the electron transport chain (ETC) and represents one of the entry enzymes of cellular respiration and contributes to the production of ATP. The NADH dehydrogenase gene was mentioned before in relation to AD. In 1992, Lin et al. identified two mutations in the mtDNA of AD patients, located in subunit 2 of the NADH dehydrogenase gene, but these results were not confirmed by other studies (Lin et al. 1992; Grazina et al. 2006). Recently, a meta-analysis was carried out by Holper et al. (2019) of both complex I and complex IV activities in many disorders, including AD. They provided strong evidence for impairments in both enzymes’ activities in the blood and some brain regions. These mitochondrial enzymes are well documented in AD and seem to be affected by amyloid β peptide (Aβ) accumulation in mitochondria, inducing neural toxicity and inhibiting mitochondrial enzymes, finally leading to mitochondrial dysfunction (Giachin et al. 2016; Coskun et al. 2012; Cenini et al. 2016). Thus, it is clear from the literature that complex I has been implicated in neurodegenerative diseases, including AD, but no previous findings were found regarding A13759G in association with AD.
The second positive association was with A5656G. This polymorphism is the characteristic SNP of sub-haplogroup U5b1. Ridge et al. (2013) showed that four clades (U5B1, U5B1B2, K1A1B, and K1A1B2A1) were associated with different endophenotypes of AD patients. Our results confirm and extend these previous studies, proving that sub-haplogroup U5b1 is probably implicated in AD. Furthermore, our findings show that the A5656G polymorphism may be the main factor in this sub-haplogroup’s involvement in AD. This SNP is located in the non-coding region separating two structural genes of tRNAs: tRNA (Ala) and tRNA (Asn). Interestingly, a similar transition in position 3302 has been reported to disturb the processing of primary transcripts, causing a functional alteration (Bindoff et al. 1993). Thus, the mechanism is still not clear, but it seems probable that this polymorphism located between two tRNA genes might cause a functional defect (Zsurka et al. 1997). This theory is plausible because the A5656G transition was found in many diseases: tubulointerstitial nephritis (Zsurka et al. 1997), occipital stroke (Finnilä, Hassinen, and Majamaa 2001), and Parkinson’s disease (Vives-Bauza et al. 2002).
An increased mutation frequency was observed in the brains of AD patients (Chang et al. 2000). Also, it has been suggested that mutations in mtDNA might change the age of AD onset and contribute to the process of neurodegeneration (Grazina et al. 2006). Unfortunately, only a few SNPs were described as being linked to AD risk; an example is the variant at position 4336 in the tRNA (Gln) gene (Shoffner et al. 1993; Egensperger et al. 1997). The associated SNPs (A5656G and A13759G) found in our study were not mentioned before as a risk factor for AD in previous studies. Since mtDNA encodes for mitochondrial subunits, transfer RNAs, and ribosomal RNAs, it is logical to think that mutations or clusters in the mtDNA might alter mitochondrial metabolism and lead to AD (Maruszak et al. 2009). However, oxidative stress can explain the high level of mitochondrial mutations.
In other studies, brains of elderly subjects and AD patients showed an increased mutation rate in comparison with young subjects, indicating that mutations in mtDNA are not specific to the pathogenesis of AD. Mutations are also observed in ageing (Lin et al. 2002), so it is unclear whether an increased mutation rate is caused by AD or ageing.
At the same time, evidence of maternal transmission of AD was mentioned in many studies. Honea et al. (2012) reported that persons with a maternal history of dementia showed an increased presence of a biomarker of AD compared with persons with paternal AD or no history of AD. Since mtDNA is maternally transmitted, it is logical to think about the implication of mtDNA in the pathogenesis of AD.
In view of all these hypotheses, many studies have been undertaken to understand the role of mtDNA in AD. Unfortunately, there are no conclusive results regarding the exact role of mtDNA in AD pathogenesis. Mutations in mtDNA can be a primary cause of AD, or the damages observed in mitochondria might be the result of AD.
The association of mutations in mtDNA with AD is still under discussion. An issue in this discussion is the potential involvement of nuclear DNA in mitochondrial variants in the pathogenicity of AD. No research has been conducted to evaluate the possibility of an association between mitonuclear genes and mtDNA in influencing the risk of AD (Andrews et al. 2019).
Finally, in the second part of our study, we found an association of the APOE e4 allele (p = 0.000014) with AD, confirming previous findings indicating that the APOE e4 allele is a major genetic risk factor for AD in Tunisia. The frequencies of observed genotypes were 4.3%, 82.5%, and 13.2% for the APOE alleles ε2, ε3, and ε4, respectively. These values are almost the same as those reported by previous studies on the APOE allele frequencies in the Tunisian population (Smach et al. 2008).
The APOE gene is the strongest genetic risk factor for AD (R 2019; Corder et al. 1993). This gene encodes the apolipoprotein E protein, which is implicated in cholesterol and lipid transport. Several hypotheses have been put forward to explain the relation between APOE and AD risk. One idea is that there is a connection between APOE and mitochondrial dysfunction. This hypothesis was based on the observation that the apolipoprotein E4 isoform is differently folded compared with other isoforms. This alternative folding makes it more susceptible to proteolysis, leading to a small peptide. The resulting fragments constitute toxic bioactive elements that enter the cytosol and lead to cell death by disturbing the mitochondrial energy balance (Mahley, Weisgraber, and Huang 2006; Swerdlow et al. 2017).
In addition, the interaction of APOE ε4 and mtDNA could be of interest for AD pathology. This idea was put forward in a cognitive study on the association of parental dementia with AD. The results revealed that non-demented APOE4 carriers with an AD-affected mother showed less memory performance than individuals with an AD-affected father with a higher rate of brain atrophy (Debette et al. 2009). This finding could point to the possibility of an interaction between APOE4 and certain mitochondrial regions or haplogroups in modulating AD risk. Indeed, several previous reports have mentioned that some mitochondrial haplogroups (U and K) seem to neutralise the harmful impact of APOE4 (Carrieri et al. 2001). One study proposed that some mitochondrial haplogroups have an influence on allele 4 of APOE, the major risk factor of AD. In particular, haplogroups K and U have been suggested to neutralise the effect of APOE4 (Carrieri et al. 2001). This hypothesis could be investigated in future studies with a larger number of samples of the Tunisian population.