Chapter 15

Transcranial photobiomodulation treats

Alzheimer’s disease in amyloid-β protein
precursor transgenic mice
Luis De Taboada1 and Michael R. Hamblin2,3
1
Chief Technology Officer, LiteCure LLC, New Castle, DE, United States, 2Wellman Center for Photomedicine, Massachusetts General Hospital,
Boston, MA, United States, 3Department of Dermatology, Harvard Medical School, Boston, MA, United States

15.1 Introduction
There is evidence suggesting that the primary mitochondrial chromophore or photoacceptor molecule for photobiomo-
dulation (PBM) is cytochrome-c-oxidase (CCO) (Wong-Riley et al., 2005; Karu, 2010). The CCO enzyme complex
contains two copper centers, CuA and CuB, with the CuA center having a broad wavelength absorption peak between
800
830 nm in its oxidized form. CCO is a terminal enzyme in the cellular respiratory chain that is located in the inner
mitochondrial membrane. It plays a central role in the bioenergetics of eukaryotic cells by moving protons across the
inner membrane and driving the formation of ATP by oxidative phosphorylation (Tuner and Hode, 2002). A possible
mechanism of action resulting in neuroprotection following transcranial photobiomodulation (tPBM), is an increase in
ATP formation (Lapchak and De Taboada, 2010), which then leads to the preservation of compromised tissue in the
ischemic penumbra. PBM can reduce apoptosis by altering mitochondrial signaling molecules, and upregulating antia-
poptotic proteins (Bcl2 and survivin) thus enhancing neuroprotection (Blivet et al., 2018) and neurorecovery (Janzadeh
et al., 2016). Since AD has been linked to mitochondrial dysfunction (Lynn et al., 2010), tPBM may be a viable treat-
ment for AD (Hamblin, 2016). Therefore, we hypothesized that the efficacy of tPBM may be mediated by enhanced
mitochondrial function (Trimmer et al., 2009).
Amyloid-β (Aβ)-containing senile plaques represent one of the neuropathological hallmarks of AD with considerable
effort, having been expended in understanding the relationship of Aβ and Aβ-containing senile plaques to AD
pathophysiology (Harrington, 2012). Much of this work has focused on the biosynthesis of Aβ and factors that influence
its production and deposition. Aβ peptides are primarily generated via internal proteolysis of its precursor, the amyloid-β
protein precursor (AβPP), giving rise to two peptides of either 40 or 42 amino acids (De Strooper and Annaert, 2000).
In addition to Aβ-containing senile plaques, a variety of neuronal cytoskeletal alterations are prominent features of AD
neuropathology. These features include hyper-phosphorylated-tau-containing neurofibrillary tangles, dystrophic neurites
present in senile plaques, and loss and deterioration of synapses (Selkoe, 1999). Whether these abnormal features are
the result of or cause of neuronal loss is still controversial (Jack et al., 2016). Regardless of the precise mechanism, this
neuronal and synaptic loss leads to cognitive decline (De-Paula et al., 2012).
Early onset autosomal dominant AD is directly linked to mutations in one of several genes: AβPP, presenilin 1
(PS1), or presenilin 2 (PS2) (Levy-Lahad et al., 1995; Lin et al., 2000). In addition, several risk factor genes, most nota-
bly the apolipoprotein E 4 allele, alter the risk for later onset AD (Strittmatter et al., 1993). It is therefore clear that
mutations or polymorphisms in several genes can lead to similar AD phenotypes (Sancesario and Bernardini, 2018).
Secretases act on APP to cleave the protein into three fragments. Sequential cleavage by β-secretase (BACE) and
γ-secretase produces the AβP peptide fragment that aggregates into clumps called plaques. If α-secretase acts on APP
first instead of BACE, no amyloid-β is formed because α-secretase recognizes a target protein sequence closer to the
cell surface than BACE. The nonpathogenic middle fragment formed by an α/γ cleavage sequence is called P3 (John,
2006). In addition, the γ-secretase enzyme (a complex of PS1 and PS2) cleaves the transmembrane domain to release
Photobiomodulation in the Brain. DOI: https://doi.org/10.1016/B978-0-12-815305-5.00015-4
© 2019 Elsevier Inc. All rights reserved. 207
208 PART | II Studies in animal models

the Aβ peptide and the carboxyl terminus fragment (Weihofen et al., 2002). Altered functions of any of these enzymes
can lead to enhanced production of Aβ peptide, which may contribute to AD pathogenesis. A number of studies have
shown that mutations in the APP gene or in presenilins result in increased β-secretase cleavage and production of both
Aβ1-40 and Aβ1-42 (Selkoe, 2001).
Transgenic (Tg) mice that overexpress mutant familial AD APP genes have contributed to an understanding of AD
pathology, and support the amyloid cascade hypothesis (Kokjohn and Roher, 2009). Although many sophisticated mice
APP models exist, none perfectly recapitulate AD cellular and behavioral pathology. The morphological resemblance to
AD amyloidosis is impressive, but fundamental biophysical and biochemical properties of the APP/Aβ produced in Tg
mice differ substantially from those of humans. The greater resilience of Tg mice in the presence of substantial Aβ
burdens suggests that levels and protein isoforms that are deleterious to human neurons, are not as noxious to these
mice. We assessed the effects of tPBM in a hAPPwt mouse model (Hook et al., 2009) by determining the effects of
tPBM on the reduction in Aβ peptide levels, and inflammatory markers, on the halting and reversal of amyloid deposi-
tion. Moreover, the key goal of AD therapy, that is, behavioral improvement was tested (De Taboada et al., 2011).

15.2 Study design

The experimental design was described in detail in our previous publication (De Taboada et al., 2011). One hundred male
AβPP transgenic mice at 3 months of age, were randomly divided into five groups. Mice received tPBM or sham treatment
three times a week for 6 months. The optical parameters were: spot size, 3 mm diameter; laser wavelength, 810 nm; peak
radiant powers of 40, 200 or 400 mW; laser modulation, either CW or pulsed with 2 ms pulse duration at 100 Hz. Group 1
was sham, group 2 was CW (40 mW), group 3 was pulsed (P1) 40 mW, group 4 was pulsed (P2) 200 mW, and group 5 was
pulsed (P3) 400 mW. Mice were tested once in the MWM between days 176 and 180. Mice were sacrificed on day 180 and
the brains were removed for analysis. The amyloid load in the brain was measured by immunohistochemistry using thin sec-
tions, levels of Aβ peptides and cytokines in cerebrospinal fluid (CSF), plasma and brain were measured by ELISA, ATP
was measured by a luciferin/luciferase assay, and oxygen consumption by mitochondrial fractions with a Clarke electrode.

15.3 Transcranial photobiomodulation improves cognitive performance as measured by

Morris Water Maze
It is important when studying new treatments in mouse models of AD to include a cognitive performance test in the
outcome measures, as previous studies relying solely on amyloid peptide or plaque measurements have not translated
into effective clinical treatments (Mehta et al., 2017).
In the present study, we used the Morris Water Maze (MWM) which is probably the most well-validated behavioral
test for spatial memory and learning that can be applied to rodent models (Edwards et al., 2014). Fig. 15.1 shows the
improvements in cognitive performance as measured by the MWM. The same pattern was evident in both MWM
outcome measures in which the pulsed light at 200 mW was the best, with the pulsed at 40 and 400 mW less effective
and the CW light least effective (but still significantly better than the sham). The improvement in time spent in target
quadrant (memory) with the P2 regimen was impressive (threefold increase).

(A) 60 (B) FIGURE 15.1 Effect of tPBM

Time spent in target quadrant on Morris Water Maze behav-
20 *** ioral studies. (A) Effect of tPBM
50 Latency time on Morris water maze latency time
(s). (B) Effect of tPBM on Morris
40 15 *** water maze time spent in the target
*** *** quadrant (s). Mean6SEM (n520)
Seconds

Seconds

*** ***P,.001, **P,.01, *P,.05.

30 *** ***
10
***
20

5
10

0 0
Sham CW P1 P2 P3 Sham CW P1 P2 P3
 Transcranial photobiomodulation treats Alzheimer’s disease in amyloid-β protein precursor Chapter | 15 209

15.4 Transcranial photobiomodulation lowers the amyloid load in brain and reduces levels
of Aβ peptides in brain, cerebrospinal fluid, and plasma
We next proceeded to measure the levels of amyloid load and the levels of Aβ peptides in brain, CSF, and plasma as
shown in Fig. 15.2. The pattern was similar for all four measures, that is, P2 , P1DP3 , CW , sham. By and large the
reductions in the brain were larger than the reductions in the CSF and in the plasma.

15.5 Transcranial photobiomodulation reduces inflammation in the brain

One important feature of AD that should be affected by any potential treatment, is the neuroinflammation that is so
characteristic of the disease progression. This inflammation is hypothesized to arise from activated microglia that are
failing to remove the accumulating amyloid plaque (Cai et al., 2014). We measured the levels of three prototypical M1
microglial inflammatory cytokines (interleukin-1beta IL-1β; (B) tumor necrosis factor alpha, TNFα; (C) transforming
growth factor beta, TGFβ) in brain samples by ELISA. The results are shown in Fig. 15.3. It can be seen that the pattern
was remarkably similar for all three cytokines. There was a large increase in the transgenic AD mice compared to their
age matched wild-type counterparts, the CW tPBM produced a modest decrease, the P1 and P3 regimens produced a
further decrease, while in every case the P2 regimen produced the largest decrease.
A three-way vicious cycle of inflammation can be formed between Aβ accumulation, activated microglia, and
microglial inflammatory mediators, which enhances further Aβ deposition and increases neuroinflammation. Since
PBM can reverse the microglial activation phenotype from M1 (cytokines) to M2 (phagocytosis), this may not only
reduce M1 inflammatory cytokines, but also allow the M2 microglia to dispose of the accumulated plaque.

(A) (B) FIGURE 15.2 Effect of tPBM on

2 300 (A) amyloid load in brain, (B) level
Amyloid load CSF Aβ of Aβ peptides in CSF, (C) level of
Aβ peptides in plasma, (D) level of
% Amyloid in brain sections

250
1.5 Aβ1-40 peptide in brain. ***P , .001,
**P , .01, *P , .05.
CSF Aβ (pg/mg)

** 200 *

**
1 *** 150
***
100
0.5
50

0 0
(C) Sham CW P1 P2 P3 (D) Sham CW P1 P2 P3
250
Plasma Aβ 2 Brain Aβ 1-40

200
β 1-40 (ng/mg)
β (pg/mg)

*** *** 1.5 ***

150
***
Plsma Aβ

*** ***
Brain Aβ

1 ***
100
***

50 0.5

0 0
Sham CW P1 P2 P3 Sham CW P1 P2 P3
210 PART | II Studies in animal models

(A) (B) (C) 500

250 IL-1β 140
TNF-α TGF-β

* 120 400
200 ***
*
100

TNF-1α (AU)
β (AU)

TGFβ (AU)
*** 300
150 80
***
IL-1β

***
***
60 *** 200
100 ***
***
***
*** 40
50 *** 100 ***
20

0 0 0
WT Sham CW P1 P2 P3 WT Sham CW P1 P2 P3 WT Sham CW P1 P2 P3

FIGURE 15.3 Effect of tPBM on (A) interleukin-1beta, (B) tumor necrosis factor alpha, (C) transforming growth factor beta, in brain samples mea-
sured by ELISA. ***P , .001, **P , .01, *P , .05.

15.6 Transcranial photobiomodulation improves mitochondrial function in the brain

One of the earliest established and most robust mechanisms of PBM was the increase in mitochondrial ATP synthesis
(Pastore et al., 1996) and a consequent increase in oxygen consumption due to respiration (Pastore et al., 1994). Since it
is accepted that an important feature of AD brains is mitochondrial dysfunction (Picone et al., 2014), it made sense to
measure ATP and mitochondrial oxygen consumption in the AD mouse brain samples after the tPBM protocols. We
only used the P2 tPBM protocol since by now, we had convinced ourselves that this was clearly the best set of para-
meters. The results are shown in Fig. 15.4.
The transgenic mice had a marked decrease in both brain ATP levels (only one-third of the WT type mice) and in
mitochondrial oxygen consumption (less than half the WT mice). However, in both cases (brain ATP and brain mito-
chondrial oxygen consumption), the P2 tPBM restored the levels to such an extent that there was no statistically signifi-
cant difference between the tPBM treated AD mice and the healthy WT mice.

15.7 Discussion
One interesting result of this study was the marked superiority of one set of tPBM parameters namely P2, consisting of
200 mW peak power (40 mW average radiant power) pulsed at 100 Hz. The biphasic dose response is well known in
PBM (Huang et al., 2009, 2011) and the present data appears to be a remarkable example of this. The P2 dose was sig-
nificantly superior to both 40 and 400 mW displaying a typical Arndt-Schulz curve. Moreover, PBM pulsed at 100 Hz
with an average power of 40 mW was better than CW with the same average power. This result agrees to some extent
with Ando et al. who found that pulsing at 10 Hz was better than CW for 810 nm laser on TBI in mice (Ando et al.,
2011). However, while, Ando et al. did not find a superiority for 100 Hz but only for 10 Hz. Ando was working with
mice suffering from TBI, while the present study used different AD mice. The convincing evidence of both improved

(A) (B) FIGURE 15.4 Effect of P2

1000 ns 160 tPBM on (A) brain ATP content
*** ** *** *** and (B) brain mitochondrial oxy-
140
gen consumption. ***P , .001,
O2 consumption (% of WT)

800
ATP 120 **P , .01, *P , .05. ns 5 not
O2 consumption
significant.
ATP (nmo/mg)

600 100

80
400 60

40
200
20

0 0
WT Sham P2 WT Sham P2
 Transcranial photobiomodulation treats Alzheimer’s disease in amyloid-β protein precursor Chapter | 15 211

cognitive performance together combined with biochemical measurement of Aβ and amyloid plaque load do suggest
that AD will be treatable with PBM.

15.8 Conclusion
Since this study was first published in 2011 (De Taboada et al., 2011), there have been a few preliminary reports pub-
lished describing case studies of human patients with AD or dementia being benefitted by PBM. Saltmarche et al.
(2017) reported a case series of five patients with mild to moderately severe dementia or possible Alzheimer’s disease
(AD) with Mini-Mental State Exam (MMSE) baseline scores of 10
24. Patients were treated with 810 nm, 10 Hz
pulsed LED devices combining transcranial plus intranasal PBM to treat the cortical nodes of the fault mode network
(DMN, bilateral mesial prefrontal cortex, precuneus/posterior cingulate cortex, angular gyrus, and hippocampus) for 12
weeks of active treatment as well as a follow-up no-treatment, 4-week period. There was significant improvement after
12 weeks of PBM (MMSE, P , .003; ADAS-cog, P , .023). Increased function, better sleep, fewer angry outbursts,
less anxiety, and wandering were reported post-PBM.
Berman et al. (2017) carried out a small pilot double blind, placebo-controlled trial (n 5 11) 6 active, 3 controls, and
2 dropouts assessing the effect of 28 consecutive daily, six minute sessions of transcranial NIR PBM using
1060
1080 nm LEDs. Results showed improvement in executive functioning; clock drawing, immediate recall, praxis
memory, visual attention, and task switching (Trails A&B) as well as a trend for improved EEG amplitude and connec-
tivity measures.
Maksimovich (2011) used a different approach. He reported a series of 46 patients aged 34
79 (average 65) with a
history of AD, who received endovascular surgery leading to transcatheter revascularization and recovery of collateral
and microvascular circulation of the brain by means of low-energy transluminal laser irradiation. Patients had a positive
outcome evidenced by a prolonged decline in dementia symptoms, and a decrease in cognitive impairment.
Taken together, the present mouse study added to the three preliminary clinical studies discussed above, suggests
that PBM may indeed be a highly encouraging treatment approach ahead of the expected “epidemic of Alzheimer’s dis-
ease” (Trempe and Lewis, 2018).

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