Alzheimer’s disease (AD) is the most prevalent cause of dementia globally, with patients suffering from progressive impairment of cognition that ultimately impedes daily activities ^[1][1]. The primary pathological hallmarks of AD are the aggregation of amyloid beta (Aβ) peptides and accumulation of hyperphosphorylated neuronal tau proteins to form neurofibrillary tangles and subsequent chronic inflammation of the brain ^[2]. Genetic factors of AD vary depending on the subtype of the disease, such as the presence of the ε4 variant of the apolipoprotein E (APOE) gene that is associated with the more common sporadic form of AD (sAD) that manifests in those over the age of 65, or mutations in the amyloid precursor protein (APP) and presenilin (PSEN) genes that bring about the early onset of Alzheimer’s ^{[2][3][4][5][6]}[3,4,5,6]. A range of causal mechanisms have been suggested to contribute to AD, including neuroinflammation ^[7], infectious agents ^[8], synaptic metabolic failure ^[9], and diabetes and obesity ^[10]. Urgent attention is required to manage, attenuate or possibly cure AD as it is poised to be the next health crisis. Numerous possible therapeutic agents have been proposed as a means to curb the extensive effects of AD.

2. Noninvasive

2.1. Photobiomodulation

Photobiomodulation (PBM), formerly known as low-level laser (light) therapy (LLLT), utilizes laser or light as a noninvasive form of treatment. Although red or near-infrared (NIR) light has been the focus of many investigations, wavelengths of light from the visible (380–720 nm) ^[11] to the infrared spectrum (600–100 nm) ^[12] have been employed as a form of phototherapy where light is absorbed by chromophores present within cells. The absorbed light activates a chain of reactions that eventually leads to the upregulation of transcription factors involved in numerous cellular processes tasked with protecting the cell. While the application of PBM to the body is a relatively easy task, application to the nervous system, especially the brain, is more challenging. Some success has been achieved when applied through a sinus ^[13] or via a transcranial approach ^[14]. PBM wavelengths can penetrate up to 5 cm depending on the selected wavelength [15,16,17,18]^{[15][16][17][18]}. Although Aβ and neurofibrillary tangles are hallmark AD pathologies, patients also exhibit mitochondrial dysfunction, deficits in energy metabolism, increased reactive oxygen species (ROS), and neuroinflammation. Preclinical as well as clinical studies have demonstrated that PBM has the capability not only to address these issues, but also exert neuroprotective effects and therefore has a potential role in preventing AD progression.

2.1.1. Mechanisms of Action

As the mitochondrial chromophore cytochrome c oxidase (CCO), otherwise known as complex IV, is one of a few proteins that absorbs light in the near-infrared spectrum and is also associated with energy metabolism, the enzyme has been shown to play a significant role in PBM. CCO exerts its action in the final step of the electron transport chain where it mediates the transfer of an electron from cytochrome c to molecular oxygen ^[19]. This process results in the increase in mitochondrial membrane potential and adenosine triphosphate (ATP) while modulating levels of reactive oxygen species (ROS) and inducing various transcription factors involved in key protein synthesis. A study evaluating the benefits of applying PBM at various wavelengths to rat postnatal visual cortex neurons, which previously were exposed to potassium cyanide, found that wavelengths of 670 nm and 880 nm were the most therapeutic ^[20]. These wavelengths coincide with the absorption range of oxidized CCO. Similarly, Wang and colleagues discovered that PBM treatment at 660 nm and 810 nm increased ATP levels and mitochondrial membrane potential whereas wavelengths of 415 nm (blue) and 540 nm (green) had an opposite effect ^[21]. Strong evidence demonstrating that PBM is capable of inducing changes in mitochondrial membrane potential, levels of ROS as well as ATP, supports the premise that CCO is involved in the underlying mechanisms of PBM [22,23]^[22][23]. Although the involvement of CCO is a prominent theory of investigations examining the effects of PBM, there is evidence suggesting that CCO may not be the key element. A study employing two genetically modified cell lines deficient in their ability to produce CCO found that both cell lines were still able to display benefits such as increased ATP synthesis and cell proliferation following PBM treatment at 660 nm ^[24]. An emerging theory also asserts the importance of water. Kim suggests the involvement of the fourth phase of water, referred to as the exclusion zone, and the nature of its interaction with hydrophilic surfaces such as biological molecules ^[25]. Similarly, Sommer and colleagues theorize that mitochondrial-bound water, in particular, is specifically significant to the mechanisms underlying PBM ^[26]. Other theories involve the action of light-gated ion channels ^[27] or propose that PBM has a systemic effect, given that studies have discovered therapeutic responses in regions of the body remote to the site of treatment. As an example, Fafara and colleagues irradiated mesenchymal stem cells in the tibia of mice and discovered a 68% reduction in Aβ deposition in the hippocampus ^[28].

2.1.2. Effects of PBM on Amyloid Beta and Tau

The body of literature on the effects of PBM in AD is limited and mainly consists of investigations in either cellular or mouse models of the disease. Considering the potential of tau to influence normal tissue, Comerota and colleagues treated 12-month-old transgenic mice with NIR light (670 nm, 4 J/cm² of light energy) for 90 s daily, 5 days/week for 4 weeks and discovered reduced tau and Aβ pathology in cortical and hippocampal synaptosomal extracts that translated to improved long-term memory in the treated animals ^[12]. These improvements were partially facilitated by activation of the heat shock and autophagy pathways ^[12], demonstrating once again that activation of clearance mechanisms may hold the key to treatment for the disease. A second study employing the more disease-aggressive 5xFAD mouse model of Alzheimer’s where plaque seeding occurs as early as two months of age also reported beneficial effects of PBM. Cho and colleagues treated 2- and 6-month-old 5xFAD mice for 20 min a day, 3 times per week for a period of 14 weeks (610 nm, 1.7 mW/cm²; 2.0 J/cm²). Following treatment, all mice were tested at 10 months of age in the Morris water maze (long-term and spatial memory), elevated plus maze (anxiety), and passive avoidance task (short- and long-term memory) ^[29]. Results demonstrated that PBM delivered at the very early stages of amyloid deposition exerts positive effects on both memory and pathology ~4.5 months following treatment. Mice treated at 2 months demonstrated near normal spatial-, short-, and long-term memory when compared to wild-type littermates and also displayed significantly reduced amyloid plaque load and microgliosis in their brains ^[29]. The results also demonstrated that treating mice at 2 months of age compared to 6 months yielded better results ^[29] but this could probably be attributed to the accumulation of amyloid from 2 to 6 months and the burden of toxicity and inflammation endured during that time. Furthermore, although levels of APP, BACE1, and neprilysin (an Aβ degrading enzyme) were unchanged, there was a significant increase of insulin-degrading enzyme (IDE), another enzyme capable of degrading Aβ. This set of results suggests that even though PBM may not prevent the formation of Aβ, treatment promotes Aβ degradation through IDE ^[29]. Changes in power densities and type of PBM doses have also been shown to differentially affect amyloid load ^[14]. Employing a wavelength of 808 ± 10 nm, De Taboada and colleagues demonstrated that pulsed treatments (100 Hz, 2 ms duration) outperformed continuous treatments in efficacy with pulsed treatments at a power density of 2830 mW/cm² at the surface of the skin (50 mW/cm² at the cortical surface), eliciting better outcomes compared to power densities of 566 mW/cm² and 5660 mW/cm² at the skin surface (10 mW/cm² and 100 mW/cm², respectively, at the cortical surface). The volume of brain amyloid was decreased by 67% at a density of 2830 mW/cm², 54% at 566 mW/cm², and 37.3% at 5660 mW/cm². Brain levels of Aβ_1–40 and the more fibrillogenic Aβ_1–42 were also significantly decreased by pulsed PBM treatments ^[14]. PBM has also been shown to be efficacious in other mouse models of disease, including the K3 tau transgenic and the APPswe/PSEN1dE9 (APP/PS1) models ^[30]. Five-month-old APP/PS1 and 7-month-old K3 mice were exposed to a 670 nm NIR light source (44 mW/cm² delivered to the skull) for a period of 90 s, 5 days/week for 4 weeks. Immunohistochemical analysis revealed significant decreases in hyperphosphorylated tau in the neocortex and hippocampus of K3 mice ^[30]. In addition to changes in tau, Purushothuman and colleagues also observed significant decreases in the oxidative stress markers 4-HNE and 8-OHDG ^[30]. PBM was also effective in regulating Aβ levels in the APP/PS1 mouse brain where plaques, gliosis, and synaptic loss was evident at around 4 months of age and cognitive decline emerged between 6 and 10 months and worsened with age. Here the researchers discovered a significant reduction in the percentage plaque burden, average plaque size, and the number of plaques in both the neocortex and the hippocampus ^[30]. Employing the TASTPM mouse, a different APP/PS1 model of AD where plaque pathology, gliosis, and cognitive impairment are observed at around 6 months of age, Grillo and colleagues delivered a 1072 nm wavelength light (pulsed at 600 Hz with a duty cycle of 300 μs, 5 mW/cm²) in 6-minute sessions for two consecutive days, bi-weekly for 5 months ^[31]. Evaluation of mice brains (n = 3) at 7 months of age revealed significant increases in the levels of a number of heat shock proteins (Hsps) and a reduction in Aβ levels in the brain. Hsps are a large family of molecular chaperones that play roles in protein maturation, refolding, and degradation. They form complexes with family members and function to prevent the misfolding of proteins, and the refolding of misfolded proteins, thereby suppressing subsequent protein aggregation and transporting misfolded proteins or aggregates to the ubiquitin–proteasome system for degradation. Considering that Alzheimer’s is a proteinopathy-related disease, studies investigating Hsps in human and mouse brains have revealed altered levels of many family members [32,33,34,35,36]^{[32][33][34][35][36]}. The ability of PBM to increase levels of Hsps reveals a functionality that may facilitate the refolding of misfolded aggregates of Aβ and tau that contribute to AD pathology and/or the targeted degradation of these aggregates via the ubiquitin–proteosome system. Although the sample size used in this experiment was very small (n = 3), the results are promising and should be further investigated. Collectively, the studies outlined above demonstrate a role for PBM in modulating levels of Aβ and tau in the brain of animal models of AD. The results of these studies are promising and although they do not outline pathways that facilitate the removal of Aβ and hyperphosphorylated tau from the brain of these animals, there are suggestions that warrant further research.

2.1.3. Effects of PBM on Neurotrophic Factors

In addition to their effects on modulating levels of Aβ and tau in the brain, PBM has also been shown to modulate levels of neurotrophic factors in both cellular and mouse models of AD. Neurotrophic factors are a family of secreted proteins that are required for directing the growth, survival, and differentiation of neurons throughout the central and peripheral nervous systems. Particularly important for brain development and growth are neurotrophins, nerve growth factor (NGF), and brain-derived neurotrophic factor (BDNF). BDNF is crucial for neuronal plasticity and is highly expressed in brain regions such as the hippocampus, hypothalamus, and cortex ^[37]. Initial studies of BDNF function found it to be a pro-survival protein, supporting neurons during bouts of chronic stress ^[38]. Subsequent studies have characterized BDNF as harboring anti-apoptotic and antioxidative properties [39,40]^[39][40]. Functions attributed to BDNF are widespread and include facilitating memory formation ^[41] and enhancing the growth and reorganization of dendritic spines in response to changing neuronal activity ^[42]. WResearchers and others have demonstrated that levels of BDNF mRNA and protein are significantly decreased in the AD brain [43,44,45,46]^{[43][44][45][46]} and have suggested that modulation of endogenous BDNF levels may represent a therapeutic avenue for AD [47,48]^[47][48]. Using low-level laser irradiation (632.8 nm, 10 mW, 12.74 mW/cm²) for 0.7, 1.25, 2.5, and 5 min, Meng and colleagues reported increased BDNF expression in cultures of Aβ-treated mouse hippocampal and APP/PS1 hippocampal neurons that was transcriptionally driven via an upregulation of cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) ^[49]. PBM was also shown to rescue Aβ-induced dendritic atrophy and neuronal death that was characterized by upregulation of a post-synaptic density protein (PSD-95) expression and the increase in length, branching, and spine density of dendrites in hippocampal neurons ^[49]. Similarly, Heo and coworkers reported increased BDNF expression in the mouse hippocampus following treatment with 660 nm LEDs at a power density of 20 mW/cm^{2 [50]}. BDNF upregulation was driven through activation of the extracellular signal-regulated kinase (ERK)/CREB signal transduction pathway ^[50]. A second study employing the identical wavelength of 632.8 nm reported activation of intracellular inositol 1,4,5-trisphosphate (IP3) receptors, the main Ca²⁺-release channels in the endoplasmic reticulum that resulted in the increase of intracellular Ca²⁺, triggering consequent activation of the ERK/CREB pathway that eventually led to the increase in BDNF expression ^[51]. Increased expression of other neurotrophic factors, including nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF), have also been demonstrated following PBM treatment [52,53]^[52][53], although these are less well studied in the realm of Alzheimer’s.

2.1.4. Systemic Effects of PBM

Studies examining the ability of NIR light to penetrate tissues, especially the skull, have raised questions on the feasibility of PBM to affect brain tissue. In one of the early studies addressing this question, Jagdeo and colleagues investigated human cadaver heads and assessed the penetration of 633 nm and 830 nm light, discovering that 11.7% of the 830 nm (3.90 mW/cm²) and only 0.7% (0.44 mW/cm²) of 633 nm light was able to penetrate both the skin and skull to reach the occipital cortex ^[54]. Additionally, it was discovered that the amount of penetration also depended on the anatomical region that was being treated, with penetrance of 830 nm irradiation dropping to 2.1% in the frontal lobes and a mere 0.9% over the temporal lobes ^[54]. Subsequent studies by Tedord and colleagues, also employing human cadavers, compared the penetration capability of 660 nm, 808 nm, and 940 nm light ^[55]. They found that 808 nm light was best at penetrating both skin and bone of the skull and discovered that this wavelength of light could reach depths in the brain of 4–5 cm ^[55], important when considering the activation of deeper brain structures such as the hippocampus. In a comparison study that evaluated skull thickness and the ability of NIR light penetration, Lapchak et al. compared the transmission of 780 nm light through the skulls of four different species and found that the mouse skull transmitted ~38% of the light, while for rat skull it was 21%, for rabbit it was 11% and for human skulls it was a mere 4% ^[56], indicating that ~95% of the signal delivered to the surface of the human skull is attenuated by skin and bone. The results of this study are important as they suggest that results of experiments performed on animals cannot be directly translated to humans due to the large variance in skull thickness, tissue morphology, and fluid characteristics. Attempts at addressing these variations have shown that other routes of delivery may also be feasible. In pioneering work on this topic, Johnstone and coworkers examined the brains of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine)-treated mice, a model of parkinsonism, following 670 nm photobiomodulatory treatment directed at a site distal from the brain ^[57]. MPTP is a prodrug to the neurotoxin MPP⁺ (1-methyl-4-pyridirium) that selectively targets dopamine (DA)-producing cells in the brain. Following administration of MPTP, which destroys dopaminergic cells in substantia nigra pars compacta (SNc), Johnstone and colleagues irradiated either the head or the body of the mice and analyzed cell survival in SNc. Results revealed that while the effects were not as robust as direct irradiation of the head, treating the body of the animals produced a significant rescue of DA-producing cells in SNc ^[57]. In a follow-up investigation, this group examined the benefits of pre-conditioning whereby 670 nm NIR light (50 mW/cm², 3 min) was applied to the dorsal surface of animals’ body immediately prior to being administered MPTP and brain tissue analyzed 6 days later ^[58]. Results revealed that preconditioning animals with PBM resulted in an 85% higher protection of dopaminergic neurons in SNc compared to saline-treated mice ^[58]. Transcriptomic analysis of midbrain dopaminergic neurons following 10 days of 670 nm NIR treatment (4 J/cm² per day) targeting the dorsum and hindlimbs, followed by MPTP administration and a 7-day survival, revealed differential regulation of a large number of transcripts ^[59]. Pathway enrichment analysis revealed significant upregulation of genes including stem-cell-related CXCR4 signaling, adipocytokine signaling, oxidative stress response pathways, and those relating to cell proliferation and migration ^[59]. This study represents one of the first previews of how PBM may exert its neuroprotective action on the brain via remote PBM, and more in-depth studies are warranted. Focusing on alternate routes of delivery, Fafara et al. targeted mesenchymal stem cells (MSC) by treating bone marrow ^[28]. They discovered that weekly treatment for 2 months improved cognitive capacity and spatial learning that was accompanied by a 68% reduction in Aβ load in the hippocampus ^[28]. Pitzschke and colleagues compared the penetration of 671 nm and 810 nm light into cadaver brain that was delivered either via a transcranial or a transsphenoidal approach ^[13]. Results revealed that the best combination for delivery to the brain was 810 nm NIR light administered via the transsphenoidal route ^[13]. Intranasal PBM has received wide attention from those concerned with diseases and therapeutics affecting the brain and has been recently reviewed by Salehpour and colleagues ^[60]. PBM has the potential to treat many pathologies that arise from AD including Aβ load, intracellular tau tangles, neuroinflammation, oxidative stress, decreased trophic factor expression, and metabolic dysfunction. An important advantage of PBM is its noninvasive approach and as such, a patient can be safely treated on multiple occasions if required. PBM can provide sustainable therapeutic benefits including the stimulation of neurotrophic factor release and induce regenerative properties intrinsic to cells. Future research should focus on harnessing the optimal benefits of PBM by establishing parameters required for targeted and even personalized therapy to the brain.