4.1.2. Penciclovir

Penciclovir is another antiviral drug that targets HSV-1 DNA replication by blocking chain elongation. Cell cultures infected with HSV-1 displayed a reduction of virus and Aβ accumulation when penciclovir was administered. This was paralleled with a reduction in β-secretase and a component of γ-secretase [112].

#### 4.1.3. Foscarnet

Foscarnet has been tested for its ability to reduce HSV-1 levels in vitro. A study found it was able to reduce Aβ accumulation, although only at higher doses. It was also unable to significantly reduce virus levels. Furthermore, foscarnet was not as effective as acyclovir or penciclovir, and hence currently is not seen as the optimal antiviral drug available for AD [112].

#### 4.1.4. Valacyclovir

Valacyclovir, an antiviral medication used in HSV-1 and HSV-2 infections, has been determined to positively impact cognition by improving visual object learning, verbal memory and working memory in patients with schizophrenia [115]. Due to its effects on working memory, its effectiveness against HSV-1 and HSV-2, and its generally safe consumption, valacyclovir has been suggested as a potential therapeutic for AD. A clinical study is currently underway in which patients that both have mild AD and tested positive for HSV-1 or HSV-2 will receive valacyclovir. The aim of the study is to determine the impact of this treatment on cognition and the accumulation of amyloid and tau [116].

#### 4.1.5. Bay 57-1293

Numerous studies have determined the antiviral agent Bay 57-1293 to be effective in combating HSV-1 [117–121]. By targeting the helicase–primase complex, Bay 57-1293 can inhibit viral DNA replication, and has been found to be more potent than acyclovir. The severity and frequency of recurring HSV was also found to be reduced by use of this drug [121]. Furthermore, it was able to decrease levels of Aβ and reduce P-tau production in Vero cells infected with HSV-1 [117].

### 4.1.6. Biflavonoids

Our lab has investigated the use of bioflavonoids, including ginkgetin, isoginkgetin and ginkgolic acid, derived from the leaves of Ginkgo biloba. The antiviral capabilities of these compounds has been well established in previous studies [122–126]. Hayashi et al. determined ginkgetin to successfully inhibit the viral replication of HSV-1, HSV-2 and the human cytomegalovirus, while also suppressing viral protein synthesis [122]. Additionally, a study by Miki et al. found ginkgetin to have anti-influenza virus activity [123]. Ginkgetin has been studied for use in AD by Zeng et al., who administered the drug to APP/PS1 transgenic mice. They observed a significant reduction in Aβ plaques and an improvement in inflammation [127]. Borenstein et al. have demonstrated the ability of ginkgolic acid to limit virus infectivity by inhibiting its fusion. The study found ginkgolic acid to be successful in inhibiting HSV-1, human cytomegalovirus and zika virus. Furthermore, it was effective in inhibiting viral protein synthesis and genome replication, in HSV-1 and human cytomegalovirus, respectively [124]. Ginkgolic acid has also demonstrated antimicrobial properties, specifically against *E. coli* and *Staphlylococcus aureus* [125]. Isoginkgetin has been shown to provide neuroprotection against the cytotoxic effects of excessive Aβ accumulation [128,129], while also having anti-microbial

and anti-fungal activity [126]. Our lab's preliminary work in testing these three compounds in AD determined their effectiveness in reducing Aβ load in vitro, further supporting their therapeutic potential in AD.

#### *4.2. Antimicrobial Drugs*

#### 4.2.1. Doxycycline

Doxycycline is a tetracycline antibiotic that has been studied for its therapeutic efficacy in AD models. Contrary to other tetracyclines, doxycycline has been determined to be safe and is able to penetrate the BBB [130], allowing it to exert its effect directly in the CNS. In vivo models, in which doxycycline was administered to mice, observed its accumulation in amyloid deposits, including Aβ plaques [131]. With respect to the production and formation of Aβ oligomers, it was observed that although doxycycline administration in transgenic mice did not cause a shift in Aβ monomers, there was a significant reduction in Aβ 18-mer levels when compared to control [132]. The same study also observed a significant memory recovery in animals that received treatment; however, there was no reduction in Aβ plaque size [132]. The paper suggested this was possibly due to the short two-month period of the study, as a previous three-month study found plaque size to be significantly reduced [132]. In respect to neuroinflammation, a reduction in microglia activation has also been associated with doxycycline administration [132]. A drosophila model, which administered doxycycline to Aβ1–42-expressing flies, observed that the treated group's locomotor deficits developed slower than the control group. The same study also observed doxycycline administration to be associated with reduced Aβ fibrilization, suggesting the production of smaller amyloid structures [133]. Another study associated doxycycline with the destabilization of Aβ fibrils [134]. Clinical trials, however, were not as successful. One study, which administered doxycycline and rifampicin, observed improvements in cognitive function, as assessed by the Standardized Alzheimer's Disease Assessment Scale–Cognitive Subscale (SADAScog) score [135]. However, a second study did not find any improvements in the cognition or function of patients with mild to moderate AD with doxycycline/rifampicin administration [136]. Further investigations would be needed to understand why the benefits seen in murine models do not translate into clinical trials.

### 4.2.2. Propranolol

Propranolol hydrochloride, an antihypertensive drug shown to have antimicrobial properties [137], has also been found to impact Aβ production. Cortico-hippocampal neuronal cultures treated with this drug manifested reduced levels of Aβ production. Furthermore, the one-month treatment of Tg2576 mice resulted in roughly a 40% reduction of Aβ1–40 and Aβ1–42 levels in the brain. When administered over a period of 6 months, Aβ peptide levels were still reduced in the brain; however, no improvement in spatial memory function was observed [138].

#### 4.2.3. Rifampicin

Rifampicin is an antibiotic derived from *Nocardia mediterranei*, which has been investigated for use in neurodegenerative diseases such as Parkinson's and AD [139,140]. Rifampicin has been found to provide neuroprotection through its anti-oxidant and anti-inflammatory properties [139,141]. Furthermore, in vitro studies found that its administration improved neuronal survival and reduced microglial activation [141]. Studies by Tomiyama et al. found rifampicin to protect neurons from cytotoxicity by scavenging free radicals [142,143]. In relation to the antimicrobial hypothesis, rifampicin has been previously studied for use in bacterial cerebral infections [144]. As rifampicin is able to cross the BBB [144], it can exert its antimicrobial effect directly in the brain. In the presence of rifampicin, a reduction of Aβ fibril formation [142] has been observed in addition to augmented Aβ clearance [145]. A study by Umeda et al., in which rifampicin was administered to APPOSK mice, found the treatment to reduce Aβ accumulation, provide synaptic protection, and reduce microglial activation [146]. Clinical studies exploring the impact of rifampicin on cognitive function have also been investigated, as mentioned in previous sections. Even with its many benefits, the oral intake of rifampicin has also been associated with liver injury in humans. To circumvent this limitation, administering rifampicin intranasally or subcutaneously has been suggested [147]. These routes of rifampicin administration have been shown to be more effective in improving memory than oral administration [147].

### 4.2.4. Gingipain Inhibitors

The use of gingipain inhibitors in AD is another approach that has been taken to alleviate the negative impact of the disease. Gingipains are virulence factors that are produced by *P. gingivalis* [148]. They are made up of a group of cysteine proteinases, specifically arginin–gingipain A, arginine–gingipain B, and lysign–gingipain [148,149]. Given the key role gingipains play in host colonization [148] and the inactivation of host defenses [150–152], they are essential for the survival and pathogenicity of *P. gingivalis*. Regarding Aβ1–42 peptide production, *P. gingivalis* infection was found to increase Aβ1–42 levels. Furthermore, incubating *P. gingivalis* with Aβ1–42 peptides led to a significant increase in *P. gingivalis* death. These two findings further support the antimicrobial hypothesis for Aβ peptides [153]. Gingipain inhibitors, such as COR286, COR271 and COR388, have been found to be effective in inducing *P. gingivalis* death and reducing the bacterial load in the brain, more so than other antibiotics, such as moxifloxacin [153,154]. In addition, COR271 was found to provide some level of neuroprotection as well [153]. The administration of gingipain inhibitors has also been associated with a decrease in host Aβ1–42 response to *P. gingivalis* infection [68].

#### *4.3. Limitations*

Even with the benefits associated with the antimicrobial and antiviral drugs listed above, insights into their mechanism of action and their impact on Aβ peptide levels are needed. A greater understanding as to whether their administration indirectly reduces the presence of Aβ peptides by reducing the viral/bacterial load on the brain, or if they act directly in reducing Aβ peptides level, is needed. If it is the latter, and the antimicrobial hypothesis for Aβ peptides holds true, their efficacy might not be as positive as hoped. In addition, it is important that the chosen antimicrobial or antiviral drug does not have any adverse effects that could take away from its benefits. For example, cefepime is an antibiotic that has shown to be able to cross the blood-brain barrier and cause neurotoxic symptoms [154].

#### **5. Conclusions and Future Directions**

The findings of the numerous studies highlighted in this review present a clear indication of the role bacteria and viruses can have in AD development. Even with this conclusion, it is clear that a specific bacteria or virus alone is not responsible for AD development, as no specific bacteria or virus has been identified to be universally present in all AD brains. Rather, a number of viruses and bacteria could exacerbate the progression of neurodegenerative diseases, either independently or along with other pathogens. By exploring the presence of multiple viruses/bacteria in AD brains, future investigations can give insights into which microorganisms are most present, and whether all AD brains have both a detected and increased level of selected bacteria/virus. The use of antiviral and antimicrobial drugs early on, while the patient is still in the presymptomatic phase of AD, could have potential effectiveness in targeting the root cause of AD pathogenesis and alleviating the viral/microbial load on the brain. Further investigations into their use in AD would give greater insight regarding their efficacy and limitations.

**Author Contributions:** U.H.I. and G.M.P. conceptualized the manuscript. U.H.I. and E.Z. wrote the manuscript. U.H.I., E.Z., and G.M.P. reviewed and edited the manuscript. All authors have read and agree to the published version of the manuscript.

**Funding:** This study was supported by the Altschul Foundation and in part by grant number P50 AT008661-01 from the NCCIH and ODS. We acknowledge that the contents of this review do not represent the views of the NCCIH, the ODS, the NIH, or the United States Government.

**Acknowledgments:** Figures were made using BioRender.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**

