s2fieldsofstudy: Biology Medicine

text:
Mechanism of Action and Clinical Efficacy of CDK4/6 Inhibitors in BRCA-Mutated, Estrogen Receptor-Positive Breast Cancers: Case Report and Literature Review

Sensitivity to endocrine therapy of patients with estrogen receptor (ER)-positive metastatic breast cancer and germline BRCA1/2 mutations is not yet fully elucidated. Furthermore, the registration trials of CDK 4/6 inhibitors in combination with endocrine therapy lacked of a pre-specified subgroup analysis in BRCA1/2 mutation carriers. We report clinical history of two patients with BRCA-mutated, ER-positive metastatic breast cancer treated with letrozole plus the CDK 4/6 inhibitor palbociclib. Biological and clinical implications of the treatment outcome observed in the two cases are discussed with the knowledge of scientific evidence to date available. Overall, biological rationale, preclinical, and clinical data support the prominent role of CDK 4/6 inhibitors plus endocrine therapy, even in combination with PARP inhibitors, in the treatment of BRCA-mutated, ER-positive breast cancers. However, the interaction between Cyclin/CDK pathway, ER and BRCA is complex and evidences reported so far, albeit reliable, await confirmation in the context of future randomized clinical trials.

BACKGROUND
Up to 10% of breast cancers (BCs) are attributed to pathogenic germline mutations in the BRCA1 and BRCA2 genes (1,2). Several studies on over 10,000 cases with BRCA-associated BCs have reported estrogen receptor (ER)-positive rates of 20 and 77%, in BRCA1 and BRCA2 mutation carriers, respectively (3).
ER-positive BCs diagnosed in BRCA1/2 mutation carriers are frequently characterized by higher tumor grade and proliferation rate than ER-positive tumors occurring in non-carriers (4). Less is known about sensitivity to endocrine therapy (ET) of patients with ER-positive metastatic BC (MBC) and BRCA1/2 mutations (5,6).
Here we report clinical history of two patients with BRCA-mutated, ER-positive MBC treated with letrozole plus palbociclib. Biological and clinical implications of the treatment outcome observed in the two cases are discussed with the knowledge of scientific evidence to date available.

CASE PRESENTATION 1
In 2012, a 59 year-old woman underwent left upper paracentral quadrantectomy for pT2N1M0, stage IIB, G3, BC [ER: 40%, progesterone receptor (PR): 0%, HER2-negative, ki67: 40%]. The patient received adjuvant chemotherapy with FEC (epirubicin, cyclophosphamide, and 5 FU) for 3 cycles and Docetaxel for 3 cycles. Then, in May 2013, she started adjuvant radiotherapy and ET with anastrozole. Due to the presence of strong family history (FH) of breast and ovarian cancer, BRCA genetic testing was performed with detection of the c.5332+1G>A germline, pathogenic BRCA1 mutation. The patient had prophylactic bilateral annessiectomy in October 2015.
In March 2016 the patient presented with sternal pain. Clinical examination revealed the presence of a chest-wall palpable mass. She had fine needle biopsy (FNB) of the sternal mass with cytological diagnosis of BC metastasis (ER: 60%, PR: 2%, HER2-negative). A total body computed tomography (CT) scan confirmed a unique metastatic bone lesion of the sternum. Anastrozole therapy was then stopped and on April 22, the patient started weekly chemotherapy with paclitaxel, which was interrupted after 2 months due to the CT evidence of an increase in size of the sternal lesion (longest diameter 67 vs. 57 mm). She was then enrolled in the experimental protocol "BRAVO" (11) and treated with the Poly(ADP-ribose) polymerase (PARP) inhibitor niraparib from August 2016 to February 2017, until the CT scan showed disease progression (best response: partial response [PR]). In view of the persistence of a single painful skeletal localization of disease, in March 2017 the patient underwent local radiotherapy and ET with fulvestrant was started concomitantly. After 2 months of therapy, the disease remained radiologically stable. In November 2017, the CT scan showed an increase of sternal lesion, and the CDK4/6 inhibitor, palbociclib, was added to ET. In June 2018, after 6 cycles of treatment, the CT scan showed stable disease (SD).
The patient is currently in good clinical condition (ECOG PS 0, no sternal pain) and continuing treatment. The latest CT scan (10 January 2019) showed a gradual reduction of the sternal lesion [best response by RECIST criteria (12): SD; Figures 1A,B]. At the last visit (April 2019), the duration of response (DOR) is 10 months.

CASE PRESENTATION 2
In August 2012, after self-detection of a left breast lump, a 32 year-old woman underwent fine biopsy of the breast nodule, which was positive for invasive ductal carcinoma. Due to the young age and the presence of a positive FH, BRCA genetic testing was performed with detection of the c.8878C>T germline pathogenic BRCA2 mutation. In October 2012 the patient underwent both left and prophylactic right mastectomy. The left breast pathology report revealed the presence of a pT1cN1M0, stage IIB, G2, BC (ER: 100%, PR: 40%, HER2 negative, ki67: 25%).
From December 2012 to March 2013, the patient received adjuvant chemotherapy with FEC for 3 cycles followed by docetaxel for 3 cycles. Then, she started adjuvant endocrine therapy with LHRH analog in combination with tamoxifen.
In June 2016, a blood exam showed an increase of CA 15-3 along with CT scan documenting suspicious chest-wall lymphnodes, liver, and bone lesions. FNB and cytology of lymphnodes confirmed the presence of BC cells (ER: 100%, PR: 35%, HER2-negative). In July 2016, the patient started first line chemotherapy with capecitabine, with good PR. However, in April 2017, the CT scan showed disease progression in lung, liver, and thoracic lymph-nodes. The patient started second-line chemotherapy with eribulin, which was interrupted after 3 cycles due to pulmonary and liver progression. From August 2017 to February 2018 the patient received 9 cycles of Carboplatin (best response: PR). The treatment was then interrupted for further disease progression (neoplastic lymphangitis with dyspnea). In April 2018, the patient began a new treatment with palbociclib plus letrozole and LHRH analog. After 5 months of treatment, she reported resolution of dyspnea with PS improvement, and a significant response was observed by CT scan [best response by RECIST critera (12)

DISCUSSION
We reported clinical history of two patients with germline BRCA1/2 mutations and refractory ER-positive MBC who achieved durable response to the combination of palbociclib and ET.
There are multiple links between ER, BRCA, and CDK pathway (Figures 2, 3). Mote et al. suggested that altered expression of PR is a phenotype associated with mutations in BRCA1 and BRCA2 genes (13). Other in vitro studies suggested that the wild-type BRCA1 gene product interacts with and suppress the activity of ER-alpha (ESR1) through a direct physical and estrogen-independent interaction between the amino-terminal region of BRCA1 and the conserved carboxylterminal activation (AF-2) domain of ER-alpha (14). BRCA1 deleterious mutations abolish or reduce BRCA1 ability to inhibit ER-alpha activity (14,15). It is therefore suggested that BRCA1 could function as a brake on ER-alpha driven proliferation and BRCA1 mutations can release that brake (16) (Figures 2, 3). Furthermore, Ma et al. showed that ER is also a target for the ubiquitin ligase activity of BRCA1 (17). Mutations of the ubiquitination sites abolish BRCA1-mediated inhibition of ERalpha activity (17). BRCA1 is also able to inhibit P300-mediated ER acetylation, which is essential for the transactivation functions of ER (18). In addition to direct suppression of ER activity, wild-type BRCA1 also inhibits aromatase expression, thereby lowering estrogen levels and further reducing ER-alpha-mediated transcription (19). Overall, the higher activity of ER-alpha in BRCA mutation carriers could represent the rationale for a better sensitivity to ET in patients with ER-positive BC and BRCA1/2 germinal mutations (Figure 4).
The overexpression of cyclin D1 is found in ∼50% of BCs (20), while amplification of its corresponding CCND1 gene has been observed in 9-30% of cases (21). Overexpression of cyclin D1 correlates with ER expression, PR expression, and luminal subtypes, with a favorable impact on overall survival (OS) in whole BC series containing all tumor phenotypes (21,22). However, in the luminal A group, high expression of cyclin D1 has been associated with shorter disease-free survival (DFS), suggesting that the prognostic role of cyclin D1 depends on the molecular subtype (22). Patients with tumors with high amplification of CCND1, which are mostly of the luminal B subtype, have also been found been to have an increased risk of disease recurrence (21,22). Cyclin D1 promotes the G 1/S-phase transition by binding and activating CDK4 and CDK6 (23), and CDK4/6 inhibitors effectively block the proliferation of sensitive cancer cells by inducing G1 cell cycle arrest (24). Cyclin D1 has also CDK-independent functions, such as ligand-dependent inhibition of the Androgen Receptor (AR) (25), and the promotion of transcription of the ER-alpha gene (26). Furthermore, Cyclin D1 stabilizes the interaction between ER and SRC-1 via direct binding to both proteins (27). ER also regulates the expression of the gene encoding cyclin D1 (28). There is therefore a positive feedback loop: activation of cyclin D1 leads to ER expression, which induces more cyclin D1 production (29). Wild-type BRCA is also involved in G1 cell cycle arrest. Aprelikova et al. showed that BRCA1 binds to hypophosphorylated RB, which interacts with the E2F transcription factor to block transcription and inhibit cell proliferation (30). In the presence of BRCA1 mutations, this antiproliferative control fails. However, in those cases, CDK4/6 inhibitors may restore the G1 arrest, preventing the cell from entering mitosis (31) (Figure 4). Somasundaram et al. also suggested that BRCA1 contributes to cell cycle arrest and tumor growth suppression through the induction of the CDK2 inhibitor p21 (32) (Figures 2, 3). CDK2 is a key bypass kinase of CDK4/6 inhibition and high mRNA expression of cyclin E1 (CCNE1), which activates CDK2, has been associated with resistance to palbociclib in the PALOMA-3 trial (33). It is interesting to note that, although the loss of BRCA1 may   induce CDK2 activity through p21 inhibition, on the other hand, CCNE1 amplification and cyclin E1 protein overexpression have been reported to be mutually exclusive with BRCA1/2 mutations (34,35).
Cyclin D1 also plays a cell-cycle-independent role in DNA repair. In response to DNA double-strand breaks (DSBs), it recruits proteins involved in the homologous recombination (HR) DSB repair pathway such as BRCA1 (36), RAD 51 (37), and BRCA2 (38). Interestingly, the recruitment itself of cyclin D1 to sites of DNA damage has been shown to be BRCA2-dependent (38) (Figures 2, 3). It is important to note that HR provides accurate recombination using a sister chromatid as a template, maintaining genomic stability. However, due to the need for a sister chromatid, HR is limited to the S-phase and G2-phase of cell cycle (39). By using CDK4/6 inhibitors, cells are arrested in G1 phase where they do not have an available sister chromatid in case of DNA damage and are dependent upon the compensatory, error-prone, non-homologous end joining (NHEJ) pathway, which is less accurate and may thus promote mutations and genomic instability (40). Furthermore, a decrease of HR activity during DNA damage may be induced by CDK4/6 inhibitors also in a cycle independent manner, due to the repression of critical factors for HR such as RAD51 (41). Therefore, in patients with HR deficiency (HRD), such as BRCA mutation carriers, who are treated with CDK 4/6 inhibitors, any DNA damage may cause cancer cell death since CDK4/6 inhibitors induce compensatory activation of the less accurate NHEJ pathway and impair recruitment of RAD51 (41) (Figure 4). These findings suggest that CDK inhibitors may exert synthetic lethal effect against BRCA-mutated, ER-positive BCs (37,38).
PARP is constituted by several enzymes, which facilitate DNA repair in pathways involving single-strand breaks (SSBs) and base excision repair (BER) (42). In the presence of a PARP inhibitor, attempted DNA repair of SSB results in DSB formation. BRCAproficient cells have the ability to repair the DSB, maintaining survival, but BRCA-deficient cells are unable to repair the accumulating DSBs which lead to cell synthetic lethality (42). The randomized, phase 3 OlympiAD and EMBRACA trials showed that, among patients with HER2-negative MBC and germline BRCA mutation, PARP inhibitors olaparib and talazoparib provided a significant progression-free survival (PFS) benefit over standard therapy (43,44). Interestingly, subgroup analysis in the OlympiAD trial showed a greater efficacy of olaparib in patients with triple-negative breast cancer (TNBC) than in ERpositive ones, and also a better outcome in BRCA1 than in BRCA2 mutation carriers (43). However, in the final analysis of the study, OS, a secondary end point, was not significantly different between treatment arms both in the whole study population and among the predefined subgroups (45). At present, several ongoing trials are evaluating the combination of CDK inhibitors with PARP inhibitors (46). The combination of dinaciclib (a CDK 1, 2, 5, 9, and 12 inhibitor) with the PARP inhibitor veliparib has shown preliminary clinical benefit in patients with breast cancer, prostate cancer, ovarian cancer, and other gynecologic malignancies (47). By blocking CDK12, CDK1, or CDK2, BRCA wild-type cancer cells turn into HR-deficient cells through inhibition of BRCA1 expression and phosphorylation (48,49) and, thus, become more susceptible to synthetic lethality induced by PARP inhibitors (46)(47)(48)(49). A phase I/II trial of olaparib, palbociclib, and fulvestrant has been recently activated in BRCAmutated patients with ER-positive, HER2-negative MBC 1 .

CONCLUSION
The Cyclin/CDK pathway has a crucial role in the regulation of cell cycle progression. In the last years, CDK4/6 inhibitors have changed the treatment landscape for ER-positive BC. The clinical reports here presented show a significant activity of CDK4/6 inhibitors in BRCA-mutated, ER-positive BC patients.
BRCA genes are involved in the regulation of DNA Repair Mechanism, but are also strategic for ER expression and function.
In the presence of BRCA mutations, which abolish or reduce BRCA-dependent ER-alpha inhibition, the use of ET could restore the brake on ER-alpha driven proliferation. Moreover, due to BRCA mutations, cell cycle arrest fails, but CDK4/6 inhibitors could restore G1 arrest. The induction of G1 cell cycle arrest can be used to manipulate the activity of DNA repair pathways, especially in HR-deficient cells. In those cells, G1 cell cycle arrest may lead to a major activity of NHEJ, with consequent genomic instability and apoptosis. 1 Olaparib, Palbociclib, and Fulvestrant in Patients With BRCA Mutationassociated, Hormone receptor-positive, HER2-negative metastatic Breast Cancer -NCT03685331. Available online at: https://clinicaltrials.gov/ct2/show/ NCT03685331.
Overall, biological rationale, preclinical, and clinical data support the prominent role of CDK 4/6 inhibitors plus endocrine therapy, even in combination with PARP inhibitors, in the treatment of BRCA-mutated, ER-positive breast cancers. However, the interaction between Cyclin/CDK pathway, ER and BRCA is complex and evidences reported so far, albeit reliable, await confirmation in the context of future randomized clinical trials.

DATA AVAILABILITY
All datasets generated for this study are included in the manuscript and/or the supplementary files.

ETHICS STATEMENT
The authors of this manuscript obtained patient consent for publication of clinical data and images. The patients' details were anonymized and signed consent forms were attached to the medical records. Due to the retrospective and non-interventional nature of the study, permission by the local ethics committee was not required.

AUTHOR CONTRIBUTIONS
AMi and TZ contributed equally to the writing of the manuscript and designed the figures. AMu and DB were involved in planning and supervised the study. SG interpreted the radiological images. MM, NN, BB, PZ, and VU reviewed and approved the final version of this work.


Photobiomodulation and Coenzyme Q10 Treatments Attenuate Cognitive Impairment Associated With Model of Transient Global Brain Ischemia in Artificially Aged Mice

Disturbances in mitochondrial biogenesis and bioenergetics, combined with neuroinflammation, play cardinal roles in the cognitive impairment during aging that is further exacerbated by transient cerebral ischemia. Both near-infrared (NIR) photobiomodulation (PBM) and Coenzyme Q10 (CoQ10) administration are known to stimulate mitochondrial electron transport that potentially may reverse the effects of cerebral ischemia in aged animals. We tested the hypothesis that the effects of PBM and CoQ10, separately or in combination, improve cognition in a mouse model of transient cerebral ischemia superimposed on a model of aging. We modeled aging by 6-week administration of D-galactose (500 mg/kg subcutaneous) to mice. We subsequently induced transient cerebral ischemia by bilateral occlusion of the common carotid artery (BCCAO). We treated the mice with PBM (810 nm transcranial laser) or CoQ10 (500 mg/kg by gavage), or both, for 2 weeks after surgery. We assessed cognitive function by the Barnes and Lashley III mazes and the What-Where-Which (WWWhich) task. PBM or CoQ10, and both, improved spatial and episodic memory in the mice. Separately and together, the treatments lowered reactive oxygen species and raised ATP and general mitochondrial activity as well as biomarkers of mitochondrial biogenesis, including SIRT1, PGC-1α, NRF1, and TFAM. Neuroinflammatory responsiveness declined, as indicated by decreased iNOS, TNF-α, and IL-1β levels with the PBM and CoQ10 treatments. Collectively, the findings of this preclinical study imply that the procognitive effects of NIR PBM and CoQ10 treatments, separately or in combination, are beneficial in a model of transient global brain ischemia superimposed on a model of aging in mice.

Disturbances in mitochondrial biogenesis and bioenergetics, combined with neuroinflammation, play cardinal roles in the cognitive impairment during aging that is further exacerbated by transient cerebral ischemia. Both near-infrared (NIR) photobiomodulation (PBM) and Coenzyme Q 10 (CoQ 10 ) administration are known to stimulate mitochondrial electron transport that potentially may reverse the effects of cerebral ischemia in aged animals. We tested the hypothesis that the effects of PBM and CoQ 10 , separately or in combination, improve cognition in a mouse model of transient cerebral ischemia superimposed on a model of aging. We modeled aging by 6-week administration of D-galactose (500 mg/kg subcutaneous) to mice. We subsequently induced transient cerebral ischemia by bilateral occlusion of the common carotid artery (BCCAO). We treated the mice with PBM (810 nm transcranial laser) or CoQ 10 (500 mg/kg by gavage), or both, for 2 weeks after surgery. We assessed cognitive function by the Barnes and Lashley III mazes and the What-Where-Which (WWWhich) task. PBM or CoQ 10 , and both, improved spatial and episodic memory in the mice. Separately and together, the treatments lowered reactive oxygen species and raised ATP and general mitochondrial activity as well as biomarkers of mitochondrial biogenesis, including SIRT1, PGC-1α, NRF1, and TFAM. Neuroinflammatory responsiveness declined, as indicated by decreased iNOS, TNF-α, and IL-1β levels with the PBM

INTRODUCTION
Aging is a multifactorial process characterized by progressive decline of physiological functions and potential development of a wide array of diseases (López-Otín et al., 2013). Cognitive impairment is one of the most important aspects of aging brains (Harada et al., 2013). Mitochondrial dysfunction negatively affects neuronal metabolism by means of oxidative stress. Neuroinflammation is active in normally aging brains and is implicated in subsequent cognitive decline (Bishop et al., 2010;Khorrami et al., 2014). As a consequence of the limited availability of aged animals, and the costs of obtaining and housing an adequate number of such animals for experimental studies, natural aging models can be prohibitive. As sufficient natural aging is lengthy, rapid access to aged animals is a limiting factor of pre-clinical research. As an alternative approach, we modeled natural aging by chronic administration of D-galactose to rodents. D-galactose is a reducing hexose sugar that enters the brain and reacts with many cellular components at high concentration, producing oxidative damage to lipids and DNA (Ho et al., 2003;Sadigh-Eteghad et al., 2017). The chronic D-galactose treatment induces changes that resemble natural aging and mimics several characteristics of brain aging (Wei et al., 2005;Sadigh-Eteghad et al., 2017). The administration to rodents has been shown to cause neurocognitive impairments, including memory deficits, increased inflammatory responses and mitochondrial dysfunction (Shan et al., 2009;Sadigh-Eteghad et al., 2017;. Cerebral ischemia is a major risk factor during aging with consequences for the occurrence and severity of cognitive impairment . Cardiovascular events such as heart attacks and strokes are more common in older individuals, and the likelihood and severity of brain damage are correspondingly higher due to mitochondrial dysfunction. Therefore, age-related structural and functional changes potentially increase the vulnerability of the brain to ischemic insults (Ueno et al., 2002). During cerebral ischemia, the reduced blood flow and the subsequent lack of oxygen, glucose, and other nutrients disturb cellular homeostasis (Chen et al., 2011). Excessive production of reactive oxygen species (ROS) in mitochondria activates signaling pathways that ultimately result in further mitochondrial dysfunction and abnormal bioenergetics (Onyango et al., 2010;Olmez and Ozyurt, 2012). Transient bilateral common carotid artery occlusion (BCCAO) is a well-established experimental approach in rodents that exploits the adverse influence of acute cerebral hypoperfusion on cognitive mechanisms (Schiavon et al., 2014).
Due to the inverse relationship between the capacity for endogenous mitochondrial biogenesis and chronological aging, the ability of the brain to recover from an ischemic insult is much reduced in older individuals (López-Lluch et al., 2008). Upstream regulators of mitochondrial biogenesis, e.g., transcription factors such as SIRT1 (silent mating-type information regulation 2 homolog 1) and PGC1-α (peroxisome proliferator-activated receptor gamma coactivator-1 alpha) play key roles in the pathophysiology of brain ischemia (Chen et al., 2011). Down-regulation of markers of mitochondrial biogenesis such as NRF1 (nuclear respiratory factor 1) and TFAM (mitochondrial transcription factor A), as well as lower numbers of mitochondria, are observed in reperfusion injury after ischemia (Liu et al., 2014). In addition, decreased cerebral blood flow (CBF) yields pro-inflammatory responses such as production of tumor necrosis factor-α (TNF-α) and interleukin (IL) IL-1β from the ischemic endothelium which leads to larger infarction volumes and exacerbates brain injury (Anrather and Iadecola, 2016). In addition, NO produced by inducible NO synthase (iNOS) in activated microglia and damaged cerebrovascular endothelium is involved in the detrimental inflammatory response to an ischemic insult (Zoppo et al., 2000). The evidence suggests that mitochondria are the key targets both of ischemic injury and brain aging (Gjedde, 2005;Reddy, 2008). Here, we exploit the knowledge that the mitochondria are targets both of aging and of ischemic insults to the brain, with a potential for development of novel therapeutic approaches based on stimulation of mitochondrial biogenesis and bioenergetics. In this study, the interventions stimulated mitochondrial biogenesis and bioenergetics by regulation of the electron transfer chain (ETC).
The first intervention of interest is an application of a low power of light. Transcranial photobiomodulation (PBM) with low-levels of red to near-infrared (NIR) light (wavelengths 600-1100 nm) has been proposed as a non-invasive method of stimulation in brain disorders (Hamblin, 2016;. The procognitive benefits of this light-based intervention have been observed in animal models of Alzheimer's disease  and aging , and in patients with acute stroke (Hamblin, 2017), traumatic brain injury (TBI) (Naeser et al., 2014;Morries et al., 2015), and dementia (Berman et al., 2017;Saltmarche et al., 2017). Absorption of light by mitochondrial complex IV, the cytochrome c oxidase (CCO) and subsequent increase in ATP production have been proposed as a main mechanism of action of PBM therapy (de Freitas and Hamblin, 2016), thought to act by augmentation of cerebral bioenergetics , inhibition of neuroinflammation (Lee et al., 2016;Salehpour et al., 2018b), and increase of antioxidant activity (Lu et al., 2017).
The second potential intervention of interest is administration of Coenzyme Q 10 (CoQ 10 ), also known as ubiquinone, that transfers electrons from complexes I and II to complex III (Dallner and Sindelar, 2000). CoQ 10 supplementation is held to improve mitochondrial function by promotion of ATP production (Hargreaves, 2014) and mitochondrial biogenesis (Noh et al., 2013;Khorrami et al., 2014). In neurons, the action of CoQ 10 has been shown to protect mitochondria and lipid membranes (Somayajulu et al., 2005), as has the beneficial effect of CoQ 10 supplementation on the inflammatory response of endothelial cells (Gao et al., 2012), and the neuroprotective properties of CoQ 10 against learning impairment in aged animals (Mcdonald et al., 2005;Shetty et al., 2013).
Here, we test the hypothesis that the added effects of senescence modeled by D-galactose in mice followed by BCCAO can be reversed by the beneficial effects on mitochondria of NIR light PBM and CoQ 10 supplementation (alone or in combination), as evidenced by the potential procognitive and neuroprotective actions of the interventions.

Animals and Experimental Groups
According to the research design, 90 adult male BALB/c mice, 8-10-weeks-old and weighing 20-25 g, were purchased from Laboratory Animal Center of Tabriz University of Medical Sciences (TUOMS) Tabriz, Iran. Mice were socially housed in standard cages (five/cage), kept under controlled conditions at the temperature of 24 ± 2 • C on a 12/12 h light and dark cycle, and fed standard pellet food with tap water ad libitum. The experiments were performed under an approved protocol of TUOMS Ethics Committee (No: IR.TBZMED.REC.1396.576) and were in accordance with NIH guidelines. After a week of adaptation, mice were randomly divided into two groups: control (n = 15) and the artificially aged (AA) (n = 75). The control group received normal saline (NS) (0.9% NaCl, 0.2 ml/mice) and the AA group received D-galactose (500 mg/kg/daily) (Sigma, St. Louis, MO, United States) by subcutaneous (s.c.) injection once per day for 6 weeks . At the end of the sixth week, mice in the AA group were divided into two groups: AA either separately (n = 15) or followed by transient global brain ischemia (GBI) (n = 60). The AA mice received sham surgery. Ischemic AA mice were then subdivided into four groups that received (I) sham PBM + vehicle gavage (AA+GBI), (II) sham PBM + CoQ 10 gavage (CoQ 10 ), (III) real PBM + vehicle gavage (PBM), and (IV) real PBM + CoQ 10 gavage (PBM + CoQ 10 ) (n = 15 per group).
We compared a vehicle-treated group of AA mice to a vehicle-treated control group (AA vs. control), and we compared a vehicle and sham-laser treated group of AA mice that we further stressed with transient GBI to the vehicle-treated group of AA mice (AA+GBI vs. AA). Three globally ischemic AA groups then received three different therapeutic interventions (CoQ 10 , PBM, or PBM+CoQ 10 ) that we compared to a globally brain ischemic group of AA mice (AA+GBI) given only sham treatments (CoQ 10 , PBM, or PBM+CoQ 10 vs. AA+GBI). Figure 1 shows the study design including procedures and time periods.

Surgery
We induced transient global cerebral ischemia with the BCCAO method as previously described (Soares et al., 2016). Briefly, animals were anesthetized with inhalation of isoflurane (4%) and intraperitoneal injection of xylazine (4 mg/kg) and maintained with 1.5% isoflurane in 70% nitrous oxide and 30% oxygen through a plastic facemask. Rectal temperature was maintained at 37 • C with a heating blanket and monitored during operation. A midline anterior incision was made in the neck to expose the both common carotid arteries. Each artery was encircled with 3-0 silk thread and the vagus nerve separated. Transient global cerebral ischemia was induced by BCCAO for 20 min using microaneurysm clips (Aesculap, Germany) and CBF reduction was confirmed by laser Doppler flowmeter (LDF) (LaserFlo, Vasamedics Inc., St Paul, MN, United States). Then, anesthesia was turned off, the skin incision was sutured, and the mice were placed in a recovery cage. For sham surgery, the AA group was exposed to the same anesthetic and surgical procedures without occlusion of the carotid arteries.

Treatments
We performed the PBM therapy with a NIR laser (Thor Photomedicine, Chesham, United Kingdom) at 810 nm wavelength, 200 mW maximum output power, and 6.66 W/cm 2 irradiance with a spot size of 0.03 cm 2 . The laser operated at FIGURE 1 | Timescale of D-galactose administration, global ischemia induction, PBM and/or CoQ 10 treatments, behavioral tasks, and sampling.
Frontiers in Cellular Neuroscience | www.frontiersin.org 10-Hz PW frequency on at 88% duty cycle. For each irradiation session a total energy of 1 J (fluence of 33.3 J/cm 2 ) was delivered to the head . For transcranial PBM therapy, each mouse was held firmly and the laser probe was positioned over the midline of the dorsal surface of the head in region between eyes and ears. The CoQ 10 was purchased from Sigma-Aldrich (United States). The CoQ 10 group received the treatment (500 mg/kg/day) via gavage (Schilling et al., 2001). CoQ10 was dissolved in soybean oil as a vehicle. The combination group received the same daily dose of CoQ 10 and treated with PBM 2 hr before the gavage.
The AA+GBI group received oral vehicle and underwent identical PBM therapy procedures except that the laser device remained inactive. All oral administrations had a volume of 8 ml/kg body weight. All treatments were applied once a day for 14 consecutive days.

Neurological Impairment
In order to evaluate neurological performance, mice were subjected to neurological severity score testing (NSS) on the day before starting the learning and memory tasks. The motor ability, balance, reflexes, and alertness of the animals were assessed as different aspects of neurological outcome (Beni-Adani et al., 2001). The scale ranges from 0 to 10. The intact control mice were given the score of zero (0), whereas a score of 10 reveals greatest neurological impairment. All behavioral testing was carried out by one person blinded to the experimental groups.

Barnes Maze Task
The spatial learning and memory abilities were evaluated using a Barnes task as described previously . The maze consisted of a black circular platform 100 cm in diameter with 20 evenly spaced holes located 3.0 cm from the perimeter. A movable rectangular escape box (20 × 15 × 5 cm) was located under the escape hole. The maze was placed in a quiet room where spatial cues (four basic shapes: triangle, square, circle, and horizontal rectangle) were located on the walls. Mice were encouraged to escape by a buzzer (80 dB white noise). The task procedure had three sessions including adaptation, training, and a probe trial, and took 5 days. On first day, each mouse was habituated to the task platform by gently guiding the mouse from the maze center to the target hole. The mouse was allowed to remain undisturbed in the escape box for 1 min after entering it. Each mouse then underwent 4 days of training with four 3-min trials per day, each separated by a 3-min interval. 24 h following the last training trial, a probe trial was performed, that is, the escape box was removed and the mouse was allowed to explore the maze freely for 3 min. Between trials, the maze was cleaned with 70% alcohol to remove odor cues. Trials were recorded using a digital camera mounted above the maze and the performance of mice was analyzed using a video tracking program Etho Vision TM (Noldus, Netherlands). The performance during the training trials was evaluated by the latency time to find the target hole. Additionally, the time spent in the target quadrant, and correct/wrong relative time (time spent around the holes) during the probe session was calculated.

Lashley III Maze
Spatial reference memory and learning were evaluated using the Lashley III maze task, which is based on navigation with visual and food cues (Matzel et al., 2003). Briefly, the lateral walls and ceiling of the maze were constructed from black or transparent Plexiglas, respectively. The maze consisted of three separable parts including start box, maze arms, and goal box. The maze arms had four lanes that were evenly spaced along the maze. The small cup containing sugar reward was located in the goal box as motivation. Before the start of the test, mice were fasted for 12 h. Mice were moved to the testing room for 30 min prior to placement in the maze. At the beginning of testing, each mouse was placed in the start box for 15 s and then the Plexiglas sliding door was opened to allow the animal to explore the four alleys of the maze for 6 min. The trial is completed when the animal has entered the goal box at the end of the maze. Mice were trained once a day for 5 days and all experimental procedures were recorded using a digital camera and analyzed by a video tracking program Etho Vision TM (Noldus, Netherlands). The learning index was measured as mean time in correct arms divided by total escape time during 5 days. The number of errors (i.e., a turn in an incorrect direction) and latency time to reach the goal box were also parameters of interest (Bressler et al., 2010).

What-Where-Which (WWWhich) Task
This task has been described in detail earlier  and is therefore described here briefly. The apparatus consisted of two different contexts, namely contexts 1 and 2, made of Plexiglas. All objects for testing were assembled from LEGO R bricks. At the beginning of the task, each mouse was habituated to each context for 5 min for 1 day. After 24 h, as an exposure session, two different objects (A and B) were first located in the context 1 and the mouse was given 3 min to explore it. Then, the mouse was returned to the cage for 30 s and the locations of the objects were switched (B and A) in the context 2. Next, the mouse was placed in the context 2 and was allowed to explore it for 3 min. The test session was carried out after a 5 min interval, when the mouse encountered two copies of one of the objects (A or B) located in one of the two contexts (1 or 2) for 3 min. Exposure and test sessions were performed in 2 trials for 2 days with new objects for each day. The total observation time (sum of time spent exploring both novel and familiar objects) and locomotor activity were videotaped and data extracted using a video tracking program Etho Vision TM (Noldus, Netherlands). The episodic-like memory was defined by the discrimination index (DI) as follows: DI = (N−F)/(N+F), where N and F represent the exploration time on the novel and familiar objects, respectively.

Sampling and Mitochondrial Isolation
Mice were sacrificed with an overdose of ketamine (100 mg/kg) and xylazine (10 mg/kg), at 24 h after the last behavioral test. The whole brain (excluding cerebellum) was quickly taken out Frontiers in Cellular Neuroscience | www.frontiersin.org from the skull and placed in ice-cold isolation buffer. Then, brain tissues were cut into small pieces and homogenized in ice-cold extraction buffer. The samples were centrifuged at 600 × g in 4 • C for 5 min. The supernatant liquid was transferred into a new tube and centrifuged at 12,000 × g in 4 • C for 15 min. The pellet was resuspended in storage buffer. Protein levels were determined by Bradford method (Bradford, 1976).

Mitochondrial ROS Levels
For evaluation of the ROS level in brain mitochondria, the fluorescent dye, dichlorohydro-fluorescein diacetate (DCFDA), was used (Novalija et al., 2003). The mitochondria were incubated with 2-µM DCFDA at 37 • C for 20 min. The fluorescence intensity was determined (λ ex = 485 nm, λ em = 530 nm) in a fluorescence microplate reader. The ROS levels were represented as fluorescence intensity and normalized to samples protein.

ATP Colorimetric Assay
All procedures were done according the colorimetric assay kit (MAK190, Sigma, United States). Briefly, the homogenized brain tissue was mixed with ATP assay buffer followed by development buffer and the absorbance was measured at 570 nm. A standard curve was used to obtain the ATP concentration and presented in units of µmol per g tissue.

Mitochondrial Activity
Mitochondrial labeling was performed using MitoTracker Probes (Cell signaling, United States). The MitoTracker Green (MTG) was directly added into mitochondrial suspension (100-400 nM) and incubated for 30 min. The fluorescence intensity was determined (λ ex = 490 nm, λ em = 516 nm) in a fluorescence microplate reader. The mitochondrial activity was represented as fluorescence intensity and normalized to sample protein content.

Statistics
Descriptive data were expressed as mean ± S.E.M. Comparison of different experimental groups was carried out by a one-way ANOVA followed by the post hoc Tukey test. All analyses were performed using Graph Pad Prism 6.01 (Graph Pad Software Inc., La Jolla, CA, United States). A p-value < 0.05 was considered statistically significant.

RESULTS
We designed the experiments to answer two main questions: Did the cerebral ischemia further exacerbate the effects of artificial aging, and did one or more of three hypothetically beneficial interventions reverse the effects of the aging exacerbated by ischemia? The design of the interventions yielded the assessments below, as applied to each group of mice.

Neurological Severity
No mice in any experimental group exhibited any behavioral change, as determined by NSS test (data not shown).

Barnes Maze Training session
The values of the animals' velocity indices revealed no statistical differences among experimental groups for all four days of the training session. The one-way ANOVA followed by Tukey test showed that AA mice delayed the finding of the escape box on the 2nd (p < 0.05), 3rd (p < 0.01), and 4th (p < 0.05) day. There were no significant difference between the effects of artificial aging with or without subsequent ischemia in terms of latency (p > 0.05). The interventions, CoQ 10 and PBM, both separately and together, significantly reduced the latency on the 3rd and 4th days of training (separately p < 0.05, together 3rd day p < 0.01, 4th day p < 0.001; Figure 2A).

Probe session
As shown in Figure 2B, the AA mice spent a significantly shorter time in the target quadrant (p < 0.01). No significant difference was observed between the aged mice with or without added ischemia in terms of time spent in the target quadrant (p > 0.05), but the subsequent treatment with CoQ 10 and PBM separately or together caused treated mice to spend more time (p < 0.05, p < 0.01, and p < 0.001, respectively).
The correct-to-wrong relative times significant differed between the AA and control groups (p < 0.001). There were no significant differences between the AA and AA+GBI groups (p > 0.05), but the CoQ 10 (p < 0.01), PBM (p < 0.05), and combination (p < 0.01) treatments significantly increased the correct-to-wrong relative times in the probe session (Figures 2C,D), although the effects of the three treatments did not differ.
The mean value of the number of errors was significantly higher in the AA group on days 2-5 days (for all, p < 0.05). There were no significant differences between the AA and AA+GBI groups in terms of the number of errors (p > 0.05). The number of errors on the 4th and 5th days of training, respectively, were decreased in CoQ 10 (p < 0.01, and p < 0.05) and PBM (for both, p < 0.05) groups. In addition, the combination group showed a significant decrease in the number of errors on the 2nd and 3rd (for both, p < 0.05), 4th (p < 0.001), and 5th (p < 0.01) days ( Figure 3B).
As shown in Figure 3C, the AA mice exhibited a lower learning index compared to control mice (p < 0.01). No significant difference was observed between the AA and AA+GBI groups in terms of learning index (p > 0.05). On the other hand, CoQ 10 (p < 0.01), PBM (p < 0.05), and combination (p < 0.001) treatments significantly increased mean values of the learning index during 5-day trials.

What-Where-Which (WWWhich) Task
There were no significant differences in locomotor activity and total observation time among groups (for both, p > 0.05) (data not shown). Artificial aging significantly impaired the DI in comparison to the control group (p < 0.001), while no significant difference was observed between the AA and AA+GBI groups (p > 0.05). The CoQ 10 , PBM, and combination groups notably increased the DI value as a recognition performance (p < 0.05, p < 0.01, and p < 0.001, respectively) (Figure 4).
in combination, significantly returned the ROS values to the AA group level (for all, p < 0.001), as shown in Figure 5A.

ATP
The AA group mice had significantly reduced ATP levels (p < 0.001), with further significant reduction in the AA+GBI group (p < 0.05), and the CoQ 10 (p < 0.05), PBM (p < 0.05), and combination (p < 0.01), treatments significantly returned the ATP levels to the AA group level (Figure 5B).

Active Mitochondria Levels
There was a lower active mitochondria level in the AA group (p < 0.01), with no further significant decline in the AA+GBI group (p > 0.05). The PBM, CoQ 10 (for both, p < 0.05), and in combination (p < 0.01) separately, markedly and equally raised the active mitochondria level (Figure 5C).
partially increased cytosolic TFAM levels (p < 0.01), this increase was much more pronounced in both PBM and combination treatment groups (for both, p < 0.001) (Figures 7A,C). Figures 7B,C, mitochondrial TFAM was decreased in the AA group (p < 0.05), whereas no significant difference was observed between the AA and AA+GBI groups (p > 0.05). However, both PBM and CoQ 10 treatments, alone or in combination, remarkably increased mitochondrial TFAM levels (for all, p < 0.001).

iNOS
The artificial aging caused a significant increase in iNOS levels (p < 0.001) that was further pronounced in the AA+GBI group compared to the AA group (p < 0.05). Both the PBM and CoQ 10 treatments, alone and in combination, significantly reversed the effect on the iNOS levels (for all, p < 0.001) (Figures 8A,D).

IL-1β
As shown in Figures 8B,D, the AA group did not show a significant increase in IL-1β levels (p > 0.05). However, the AA+GBI group displayed significantly elevated IL-1β levels compared to the AA group (p < 0.05). On the other hand, the CoQ 10 (p < 0.001), PBM (p < 0.001), and combination (p < 0.01) treatments showed significantly reduced IL-1β expression levels.

TNF-α
As shown in Figures 8C,D, a remarkable increase was observed in cerebral TNF-α levels in the AA group (p < 0.001). Also, a strong increase in TNF-α levels was observed in the AA+GBI group compared to the AA group (p < 0.05). However, both PBM and CoQ 10 treatments, alone or in combination, significantly decreased TNF-α expression levels (for all, p < 0.001).

DISCUSSION
Here, we demonstrated significant and substantial improvements in neurobehavioral and molecular variables, including cerebral bioenergetics and mitochondrial biogenesis, as well as the neuroinflammatory response to artificial aging, in mice subjected to a model of transient global ischemia, by the PBM or CoQ 10 treatments, or both.
Impaired mitochondrial function and inflammatory responses are considered crucial steps in the mechanisms involved in the model of brain aging induced by D-galactose (Shan et al., 2009;Kumar et al., 2010). In previous studies, aged animals were shown to be more sensitive to cerebral injury induced by an ischemic event (Canese et al., 1998;Xu et al., 2008), and transient BCCAO is known to induce an ischemia-reperfusion injury associated with oxidative damage to cortical neurons (Yabuki and Fukunaga, 2013).
Mitochondria are the principal organelles responsible for ATP generation and neuronal energy homeostasis (Onyango et al., 2010). Injured cerebrovascular endothelium in contrast is considered to be both source and target for ROS (Strasser et al., 1997). Age-related over-production of ROS results in endothelial apoptosis through oxidation of mitochondrial proteins and damage to and mutations in mitochondrial DNA (Dai and Rabinovitch, 2009). Likewise, a large body of evidence demonstrates that increased mitochondrial ROS generation follows chronic administration of D-galactose (Pratchayasakul et al., 2017), simulating the ROS generated in aging brain. Furthermore, transient reductions of CBF can deprive the brain of O 2 and glucose, further exacerbating mitochondrial dysfunction (Sakoh et al., 2000).
Cerebral ischemia is prevalent in the aged population . In addition, susceptibility to oxidative stress caused by ischemic injury typically is higher in the aging brain compared to the young healthy brain (Lucas and Szweda, 1998). Therefore, oxidative damage induced by ischemic brain insults in the aged population plays a cardinal role in the mitochondrial dysfunction and subsequent cognitive decline in these subjects (Fukui et al., 2002).
Based on the preliminary study using 810 nm laser light in BALB/c mice , the fluence that reaches the cortical surface is only 8 J/cm 2 , when 33.3 J/cm 2 of laser fluence is delivered to the scalp. In terms of light penetration through the brain tissues of BALB/c mice, it has been reported that approximately 3% of 810 nm laser photons reach a depth in the brain of 5 mm, the distance from skull surface to substantia nigra compacta (SNc) region (Reinhart et al., 2017). Therefore, it could be presumed that in this study 0.25 J/cm 2 of NIR light reaches the brainstem, which is in the biostimulatory range for PBM therapy (Sharma et al., 2011;Moro et al., 2014).
A main mechanism of the beneficial effect of NIR PBM therapy relies on the improvement of mitochondrial function by direct stimulation of the ETC (Salehpour et al., 2018d). In this context, Lapchak and de Taboada (Lapchak and De Taboada, 2010) reported an increase in ATP content following 808 nm laser therapy and proposed this mechanism as a possible explanation for the reduction of ischemic tissue and the behavioral FIGURE 5 | (A) DCFDA intensity as mitochondrial ROS levels, (B) ATP content, and (C) MitoTracker Green (MTG) intensity as mitochondrial activity in brain tissue of different groups. Each bar represents the mean ± SEM, (n = 8). * * p < 0.01 and * * * p < 0.001 compared with the NS (control). $ p < 0.05 and $$ p < 0.01 compared with the AA. # p < 0.05, ## p < 0.01, and ### p < 0.001 compared with PBM-AA + GBI. AA, artificially aged; CoQ 10 , Coenzyme Q 10 ; GBI, global brain ischemia; NS, normal saline; PBM, photobiomodulation; Veh, Vehicle.
improvements following experimentally induced stroke. Previous experiments with specific cognitive impairment models have shown that NIR PBM therapy can improve mitochondrial functions by increasing ATP  and mitochondrial membrane potential (MMP) , and decreasing the over-production of ROS Salehpour et al., , 2018c).
In the current study, NIR PBM therapy significantly affected mitochondrial dysfunction induced by global ischemia and D-galactose administration, as shown by increased ATP synthesis and suppressed ROS production. To enhance the therapeutic efficiency of PBM, we combined PBM with CoQ 10 , a critical cofactor in the mitochondrial ETC known to improve mitochondrial function. Four weeks of CoQ 10 supplementation exerted neuroprotection by decreasing the infarct volume of an ischemic hemisphere in aged mice (Li et al., 2007).
Oral administration of CoQ 10 for 30 days improved brain bioenergetics and reduced the effects of transient cerebral ischemia on mitochondrial function as indicated by increased O 2 consumption and ATP production (Horecky et al., 2011). It is known that CoQ 10 acts as a potent free radical scavenger against cerebral ischemia-induced oxidative damage (Ostrowski, 2000). Thus, our results, along with others, confirm that CoQ 10 supplementation rescues mitochondrial function and prevents brain damage produced by a model of cerebral ischemia applied to a model of aging in mice.
ATP levels in a mouse model of TBI. However, results from ROS levels did not show any significant differences among combination and separate treatment groups .
Although an exact understanding of the cellular mechanisms underlying the suppression of cerebral overproduction of ROS after PBM therapy is still missing, the upregulation of total antioxidant capacity and some individual antioxidant enzymes [such as superoxide dismutase (SOD) and glutathione peroxidase (GPx)] may contribute to the beneficial effects (Lu et al., 2017;Salehpour et al., 2018c). Besides antioxidants, SIRT1 is linked to the oxidative stress response in cerebral ischemia (Raval et al., 2008). Cellular defenses attenuate mitochondrial oxidative damage, via activation of SIRT1 in cerebral ischemic injury (Yang et al., 2015). In addition, SIRT1 directly affects PGC1α activity via phosphorylation and deacetylation (Cantó and Auwerx, 2009). PGC1-α is a transcriptional coactivator that upregulates the aforementioned antioxidant enzymes, SOD and GPx (St-Pierre et al., 2003). Studies have shown a key role of PGC1-α-mediated ROS metabolism in cerebral ischemia (Chen et al., 2011). In this regard, a rise in PGC1-α expression level has been proposed as a protective mechanism against ROSmediated neuronal death (St-Pierre et al., 2006). The antioxidant effect of alpha-lipoic acid through the up-regulation of SIRT1dependent PGC1-α expression has also been reported in a rat cerebral ischemia model (Fu et al., 2014). In our study, PBM and/or CoQ 10 significantly increased SIRT1 and PGC1α expression, and it seems that activation of SIRT1/PGC1-α pathway could be another possible explanation for observed reduction in ROS (Figure 9).
Total mitochondrial contents and the integrity of mitochondrial DNA both are adversely affected by cerebral ischemia because of the increase of oxidative stress (Chen et al., 2011). Mitochondrial biogenesis alters the size and number of FIGURE 7 | (A) Cytosolic and (B) mitochondrial TFAM levels in brain tissue of different groups. Each bar represents the mean ± SEM, (n = 6). * p < 0.05 and * * p < 0.01 compared with the NS (control). ## p < 0.01 and ### p < 0.001 compared with AA + GBI. (C) Representative images of cytosolic and mitochondrial TFAM protein levels detected by western blot. The GAPDH and Cyt. c were used as cytosolic and mitochondrial internal controls, respectively. AA, artificially aged; CoQ 10 , Coenzyme Q 10 ; Cyt. c, cytochrome c; GBI, global brain ischemia; NS, normal saline; PBM, photobiomodulation; TFAM, mitochondrial transcription factor; Veh, Vehicle.
individual mitochondrial units. Another essential function of PGC1-α, as a master activator of mitochondrial biogenesis, is regulation of mitochondrial content via transcription factors such as NRF1 and NRF2, which in turn upregulate mitochondrial gene expression, and TFAM can affect nuclear DNA (Hock and Kralli, 2009). For instance, down-regulation of PGC1-α in aluminum-treated rats along with a decrease of NRF1 and TFAM (which act downstream from PGC1-α) resulted in decrease of mitochondrial DNA copy number and mitochondrial abundance in the hippocampus and corpus striatum regions of the brain (Sharma et al., 2013). Moreover, SIRT1 could directly interact with PGC-1α and increase its activity and expression, leading to upregulation of mitochondrial gene expression (Nemoto et al., 2005). Therefore, activation of mitochondrial biogenesis, and thus the level of mitochondrial activity in neurons, has substantial therapeutic potential since it may improve cerebral bioenergetics in both ischemic brain insults and in general aging. Although activation of mitochondrial biogenetic regulators after PBM therapy has been reported in muscle (Nguyen et al., 2014) and mesenchymal stem cells (Yin et al., 2017), there is no reported evaluation of stimulatory effects of NIR PBM therapy on neuronal mitochondrial biogenesis in the cerebral ischemic aging model. It has been demonstrated that 4 days of NIR laser irradiation (with dual wavelengths of 808+980 nm) can induce mitochondrial biogenesis by elevation of SIRT1 and PGC-1α in C 2 C 12 muscle cells (Nguyen et al., 2014). Likewise, 660 nm light has been shown to increase the expression of mitochondrial biogenesis-associated genes including PGC-1α, NRF1, and TFAM in mesenchymal stem cells at 24 h post-irradiation (Yin et al., 2017). Increased transcription of NRF1 and TFAM through the upregulation of SIRT1 and PGC-1α expressions has also been FIGURE 8 | (A) The iNOS, (B) IL-1β, and (C) TNF-α levels in brain tissue of different groups. Each bar represents the mean ± SEM, (n = 6). * * * p < 0.001 compared with the NS (control). $ p < 0.05 and $$$ p < 0.001 compared with the AA. ## p < 0.01 and ### p < 0.001 compared with AA + GBI. (D) Representative images of iNOS, IL-1β, and TNF-α protein levels detected by western blot. The GAPDH was used as an internal control. AA, artificially aged; CoQ 10 , Coenzyme Q 10 ; GBI, global brain ischemia; IL-1β, interleukin-1β; iNOS, inducible NO synthase; NS, normal saline; PBM, photobiomodulation; TFAM, mitochondrial transcription factor; TNF-α, tumor necrosis factor-α; Veh, Vehicle.
The present data showed that both PBM and/or CoQ 10 could remarkably activate expression of genes, which are involved in mitochondrial biogenesis. Based on the findings from this preclinical research, we suggest that stimulation of the SIRT1/PGC1-α/NRF1/TFAM signaling pathway by NIR PBM and CoQ 10 could regulate neuronal mitochondrial biogenesis, thereby providing neuroprotection against delayed cell death after cerebral ischemia, especially in aged brains (Figure 9). Taken together, mitochondrial function is linked to mitochondrial content that potentially can be improved by PBM therapy. Increased mitochondrial activity in the brain following 810 nm laser irradiation has been reported by this laboratory in animal models of cognitive impairment (Salehpour et al., , 2018c, in line with the present results. Likewise, 2 weeks of CoQ 10 treatment notably augmented the mitochondrial activity. Hence, the findings provide evidence that stimulation of neuronal mitochondrial biogenesis may be one of the key mechanisms for NIR PBM and CoQ 10 to increase mitochondrial content and/or function, thus, attenuating cognitive impairment in a model of global cerebral ischemia superimposed on a model of aging. It is believed that microglial cells activated in response to brain ischemic insults exacerbate cerebral injury by releasing pro-inflammatory TNF-α and IL-1β (Lakhan et al., 2009). The accelerated proliferation of glial and endothelial cells after an ischemic insult results in rapid infarct development in senescent animals that display age-dependent ischemic outcomes (Popa-Wagner et al., 2007). Decreased levels of TNF-α Lu et al., 2017;Yang et al., 2018) and IL-1β (Moreira et al., 2009;De Taboada et al., 2011;Zhang et al.,  2014; Lee et al., 2017;Lu et al., 2017) have been reported in specific animal brain injury models following transcranial PBM therapy. Pre-treatment with NIR PBM also resulted in significant reduction of cerebral TNF-α and IL-1β levels in ischemic mice (Lee et al., 2016). In accordance with the previous reports, our results showed increased inflammatory responses in a model of concurrent global ischemia superimposed on a model of aging in mice, while both PBM and/or CoQ 10 partially, but significantly, attenuated the expression levels of TNF-α and IL-1β. Besides the proinflammatory cytokines, increased activity of iNOS in glia and infiltrating neutrophils leads to excessive amounts of free radicals following ischemic injury (Lakhan et al., 2009). The NO produced by iNOS within microglia and cerebrovascular endothelium is also involved in the deleterious inflammatory response occurring after ischemia (Zoppo et al., 2000). In vitro (Yang et al., 2010) and in vivo (Leung et al., 2002;Lee et al., 2016) studies support the idea that the anti-neuroinflammatory effects of transcranial PBM therapy may at least partly be due to its ability to modulate iNOS activity. In addition, CoQ 10 treatment has been reported to attenuate hippocampal TNFα levels in a pentylenetetrazole-induced cognitive dysfunction model (Bhardwaj and Kumar, 2016), and this finding is in good agreement with our data (Figure 9). Moreover, a study by Tsai et al. (Tsai et al., 2012) showed that CoQ 10 could protect human endothelial cells from oxidative stress via decreasing iNOS expression.
To test whether PBM and/or CoQ 10 protected against global ischemia-induced cognitive impairment performance in our aged-mouse model, we evaluated the animals in a test of spatial and episodic-like memories. Since the hippocampus plays a key role in the consolidation of memories related to spatial navigation, we subjected mice to the Barnes and Lashley III mazes as hippocampus-dependent spatial learning and memory tasks. The data showed that administration of D-galactose and global ischemia induction together significantly impaired spatial learning and memory, which is in line with previous reports (Xu et al., 2000;Sadigh-Eteghad et al., 2017;. On the other hand, 2 weeks of PBM and/or CoQ 10 treatments significantly reversed this deficiency. The procognitive benefits of NIR PBM therapy to spatial memory have been shown with the Morris water maze Dong et al., 2015) and Barnes maze (Lu et al., 2017;Salehpour et al., , 2018c in different animal models. It is well established that the hippocampal bioenergetic capacity and neuronal survival are linked to spatial memory and learning (Sadowski et al., 2004).
In this study, we tentatively conclude that augmentation of brain bioenergetic capacity and the accompanying neuroprotective effects were reflected in improved learning and memory performances in both NIR PBM and CoQ10 treated mice. We observed that NIR PBM therapy notably improved the episodic memory of the D-galactose-treated mice in the BCCAO model of cerebral ischemia. This result is in line with previous reports of Lu et al. (Lu et al., 2017) and , 2018c who demonstrated enhanced recognition memory after transcranial 810-nm laser therapy. Moreover, CoQ 10 supplementation significantly rescued recognition memory as indicated by higher DI which is in line with other work (Llewellyn et al., 2015).
Since both chronic injection of D-galactose and subjection of animals to global cerebral ischemia result in systemic damage to the brain and nervous system, we examined the whole brain for possible neurochemical alterations. Furthermore, to test whether treatments protected against global ischemia-induced cognitive decline in the aged animal model, we subjected mice to the Barnes maze and Lashley III maze (as hippocampus-dependent spatial learning and memory tasks) and the WWWhich task (as a prefrontal cortex-dependent episodic memory task).
We note that the Red/NIR light propagation in the brain raises an important issue for purposes of treatment. Lambert-Beer's law explains attenuation of photon propagation in biological media. Most recently, we showed that approximately 1% of the light incident onto the scalp surface reaches a 1-mm depth from the cortical surface, penetrating the dorsal hippocampus of the mouse, when a laser beam is focused on the bregma, (Salehpour et al., 2018a). Therefore, in the current study, we assumed that the cortical neurons and upper layers of the hippocampus received more photons than deeper structures (i.e., thalamus and hypothalamus). Given this, we also assumed that the neurochemical effects would be stronger if only the hippocampus had been sampled. However, further studies are needed to gain more insight into specific brain regions (i.e., hippocampus, prefrontal cortex, and amygdala) that are affected by PBM therapy of cognitive impairment.
Although the administration of CoQ 10 affects brain function systemically and globally, the upper cortical layers are mainly affected by transcranial PBM therapy due to the aforementioned biophysical properties. Hence, the restricted light penetration is a potential interpretative limitation of the studies (brain and non-brain tissues) of possible synergistic effects of the combination of PBM therapy and pharmacotherapy strategies.

CONCLUSION
We conclude that NIR PBM combined with CoQ 10 had multiple mechanisms of action, targeting mitochondrial bioenergetics and biogenesis as well as anti-neuroinflammation in this model of ischemic brain damage in AA animals. The present study suggests that the PBM and CoQ 10 treatments, alone or in combination, have procognitive effect in ischemic damage to aged brains of mice. It is noteworthy that the translation of the concepts of the current study in terms of models and conditions into humans is challenging. However, considering the number of elderly patients that sustain significant brain damage following a heart attack or ischemic stroke each year, it makes sense to test this relatively inexpensive and completely non-hazardous intervention (or combination of interventions) in humans. Further complementary preclinical and clinical studies are warranted for clarification of other potential applications in this area.

AUTHOR CONTRIBUTIONS
FS, FF, ME, JM, PK, and SR performed the experiments, interpreted the results, and wrote the first draft manuscript. SS-E and MF designed the experiments. SS-E, MH and AG critically interpreted data and critically revised and approved the manuscript.


The lhfpl5 ohnologs lhfpl5a and lhfpl5b are required for mechanotransduction in distinct populations of sensory hair cells in zebrafish

Hair cells sense and transmit auditory, vestibular, and hydrodynamic information by converting mechanical stimuli into electrical signals. This process of mechano-electrical transduction (MET) requires a mechanically-gated channel localized in the apical stereocilia of hair cells. In mice, lipoma HMGIC fusion partner-like 5 (LHFPL5) acts as an auxiliary subunit of the MET channel whose primary role is to correctly localize PCDH15 and TMC1 to the mechanotransduction complex. Zebrafish have two lhfpl5 genes (lhfpl5a and lhfpl5b), but their individual contributions to MET channel assembly and function have not been analyzed. Here we show that the zebrafish lhfpl5 genes are expressed in discrete populations of hair cells: lhfpl5a expression is restricted to auditory and vestibular hair cells in the inner ear, while lhfpl5b expression is specific to hair cells of the lateral line organ. Consequently, lhfpl5a mutants exhibit defects in auditory and vestibular function, while disruption of lhfpl5b affects hair cells only in the lateral line neuromasts. In contrast to previous reports in mice, localization of Tmc1 does not depend upon Lhfpl5 function in either the inner ear or lateral line organ. In both lhfpl5a and lhfpl5b mutants, GFP-tagged Tmc1 and Tmc2b proteins still localize to the stereocilia of hair cells. Using a stably integrated GFP-Lhfpl5a transgene, we show that the tip link cadherins Pcdh15a and Cdh23, along with the Myo7aa motor protein, are required for correct Lhfpl5a localization at the tips of stereocilia. Our work corroborates the evolutionarily conserved co-dependence between Lhfpl5 and Pcdh15, but also reveals novel requirements for Cdh23 and Myo7aa to correctly localize Lhfpl5a. In addition, our data suggest that targeting of Tmc1 and Tmc2b proteins to stereocilia in zebrafish hair cells occurs independently of Lhfpl5 proteins.

1
Abstract 17 Hair cells sense and transmit auditory, vestibular, and hydrodynamic information by converting 18 mechanical stimuli into electrical signals. This process of mechano-electrical transduction (MET) 19 requires a mechanically-gated channel localized in the apical stereocilia of hair cells. In mice, lipoma 20 HMGIC fusion partner-like 5 (LHFPL5) acts as an auxiliary subunit of the MET channel whose 21 primary role is to correctly localize PCDH15 and TMC1 to the mechanotransduction complex. 22 Zebrafish have two lhfpl5 genes (lhfpl5a and lhfpl5b), but their individual contributions to MET 23 channel assembly and function have not been analyzed. 24 Here we show that the zebrafish lhfpl5 genes are expressed in discrete populations of hair cells: lhfpl5a 25 expression is restricted to auditory and vestibular hair cells in the inner ear, while lhfpl5b expression 26 is specific to hair cells of the lateral line organ. Consequently, lhfpl5a mutants exhibit defects in 27 auditory and vestibular function, while disruption of lhfpl5b affects hair cells only in the lateral line 28 neuromasts. In contrast to previous reports in mice, localization of Tmc1 does not depend upon Lhfpl5 29 function in either the inner ear or lateral line organ. In both lhfpl5a and lhfpl5b mutants, GFP-tagged 30 Tmc1 and Tmc2b proteins still localize to the stereocilia of hair cells. Using a stably integrated GFP-31 Lhfpl5a transgene, we show that the tip link cadherins Pcdh15a and Cdh23, along with the Myo7aa 32 motor protein, are required for correct Lhfpl5a localization at the tips of stereocilia. Our work 33 corroborates the evolutionarily conserved co-dependence between Lhfpl5 and Pcdh15, but also reveals 34 novel requirements for Cdh23 and Myo7aa to correctly localize Lhfpl5a. In addition, our data suggest 35 that targeting of Tmc1 and Tmc2b proteins to stereocilia in zebrafish hair cells occurs independently 36 of Lhfpl5 proteins. 37 This is a provisional file, not the final typeset article 2 Introduction 38 The mechano-electrical transduction (MET) complex of sensory hair cells is an assembly of proteins 39 and lipids that facilitate the conversion of auditory, vestibular and hydrodynamic stimuli into electrical 40 signals. Our current understanding is that the proteins of the MET complex consist of the tip link 41 proteins cadherin 23 (CDH23) and protocadherin 15 (PCDH15) at the upper and lower ends of the tip 42 link respectively (Kazmierczak et al., 2007), the pore-forming subunits transmembrane channel-like 43 proteins TMC1 and TMC2 (Kawashima et  complex has also been reported (Ge et al., 2018). 63 A precise role for LHFPL5 has yet to be defined. One current hypothesis is that LHFPL5 acts as an 64 auxiliary subunit of the MET channel to stabilize the complex, similar to TARPs and the gamma 65 subunits of voltage-gated calcium channels. In mouse cochlear hair cells, PCDH15 and LHFPL5 66 require each other for stable localization at the tips of stereocilia (Mahendrasingam et , what are the molecular requirements for targeting Lhfpl5a to the hair  85  bundle? 2) Is the Lhfpl5-dependent targeting of Tmc1 to the hair bundle an evolutionarily-conserved  86 aspect of their interaction? 3) lhfpl5a mutants have defects in auditory and vestibular behaviors, yet 87 sensory hair cells of the lateral line are unaffected (Nicolson et al., 1998). What is the genetic basis for 88 the persistence of lateral line function in lhfpl5a mutants? 89 In this work, we report that teleost fish have two lhfpl5 genes, lhfpl5a and lhfpl5b. The zebrafish lhfpl5 90 ohnologs are expressed in distinct populations of larval sensory hair cells: lhfpl5a in the auditory and 91 vestibular system; lhfpl5b in the hair cells of the lateral line organ. Their divergent expression patterns 92 explain why lateral line hair cells are still mechanically-sensitive in lhfpl5a mutants. CRISPR-Cas 9 93 knockout of lhfpl5b alone silences the lateral line organ but has no effect on otic hair cell function. procedures (Westerfield, 2000). 110

Mutant and transgenic fish lines 111
The following zebrafish mutant alleles were used for this study: LSM 700 or LSM 800 laser scanning confocal microscopes. Z-stacks were analyzed using ImageJ 152 (Schneider et al., 2012). All related control and experimental images were adjusted equally for 153 brightness and contrast in Adobe Photoshop CC. 154

CRISPR-Cas9 knockout of lhfpl5b 155
An sgRNA targeting the lhfpl5b sequence 5'-CAACCCAATCACCTCGGAAT-3' was synthesized 156 essentially as described (Gagnon et al., 2014). For microinjection, 1 µg of sgRNA was mixed with 1 157 ug of Cas9 protein and warmed to 37ºC for 5 minutes to promote the formation of the Cas9-sgRNA 158 complex. Approximately 2 nl of this solution was injected into wild type embryos at the single-cell 159 stage. Efficacy of cutting was determined in two ways: 1) Single larvae genotyping -Amplification of 160 the target genomic region by PCR and running the products on 2.5% agarose gels to assay for disruption 161 of a homogenous amplicon. 2) Assaying for disruption of the mechanotransduction channel in lateral 162 line hair cells by FM 1-43 dye uptake. Larvae displaying a non-wild type pattern of FM 1-43 dye uptake 163 were raised to adulthood. Progeny from in-crosses of F0 adults were exposed to FM 1-43 dye to identify 164 founders. By outcrossing founder adults, we established a line of fish carrying a 5 base pair deletion in 165 the lhfpl5b coding region that causes a S77FfsX48 mutation (lhfpl5b vo35 ). This mutation disrupts the 166 protein in the first extracellular loop and deletes the final three of four transmembrane helices in 167 Lhfpl5b. 168

Acoustic startle response 169
Quantification of the larval acoustic startle response was performed using the Zebrabox monitoring 170 system (ViewPoint Life Sciences, Montreal, Canada) as previously described (Maeda et al., 2017) with 171 the following modifications. Each trial included six larvae which were subjected to two or three trials 172 of 6 acoustic stimuli. For each individual larva, the trial with best AEBR performance was used for 173 quantification. Positive responses where spontaneous movement occurred in the second prior to the 174 stimulus were excluded from analysis. Trials where spontaneous movement occurred for more than 6 175 of the 12 stimuli were also excluded from analysis. 176

Microphonics 177
We performed the microphonic measurements as previously described (Pacentine and Nicolson, 2019). 178 In brief, we anesthetized 3 dpf larvae in extracellular solution (140 mM NaCl, 2 mM KCl, 2 mM CaCl2, the amplitude from base-to-peak of the first peak. Larvae were genotyped as described above. This is a provisional file, not the final typeset article were fixed in 4% paraformaldehyde at 5 dpf. In experiment 2, larvae (n = 6 each genotype) were imaged 205 immediately. lhfpl5a tm290d/+ fish were in-crossed to produce wild type and lhfpl5a tm290d siblings. Larvae 206 were sorted based on the auditory and vestibular defects associated with the lhfpl5a tm290d homozygous 207 mutants. Larvae were labelled with FM 1-43 (n = 6 each). For all experiments, specimens were 208 mounted in low melting point agarose and the L1, MI1, and O2 neuromasts were imaged on a Zeiss 209 LSM 800 confocal. Cell counts were performed using the Z-stack data. 210

Statistics 211
Statistical analyses were done using the R stats package in RStudio (R Core Team, 2019; RStudio 212 Team, 2018). P-values of less than 0.05 were considered to be statistically significant. Plots were made 213 with ggplot2 (Wickham, 2016). 214

Data availability 215
The raw data supporting the conclusions of this manuscript will be made available by the 216 corresponding author, without undue reservation, to any qualified researcher. 217 4 Results 218

Teleost fish have ohnologous lhfpl5 genes. 219
Genes that have been duplicated as a result of a whole genome duplication (WGD) event are known as 220 ohnologs (Ohno, 1970;Wolfe, 2000). Due to the teleost-specific WGD, it is not uncommon to find 221 ohnologous genes in teleost fish where other vertebrate classes possess a single gene. The Danio rerio 222 (zebrafish) genome contains two lhfpl5 genes: lhfpl5a (ENSDARG00000045023) and lhfpl5b 223 (ENSDARG00000056458). To determine if lhfpl5 duplication is general to the teleost lineage, we 224 queried either GenBank or Ensembl databases to collect Lhfpl5 protein sequences for humans, mice, 225 chick, frogs, and representative sequences from 14 phylogenetic orders of ray-finned fish 226 (Actinopterygii), including 13 orders from the Teleostei infraclass and one from the Holostei infraclass 227 (Supplemental Table 1). The latter (Spotted gar, Lepisosteus oculatus) is commonly used to infer the 228 consequences of the teleost WGD, since the Holostei and Teleostei infraclasses diverged before the 229 teleost WGD (Braasch et al., 2016). Phylogenetic analysis of the Lhfpl5 protein sequences supports 230 the idea that duplicate lhfpl5 genes originated from the teleost WGD event ( Figure 1A). In all 13 teleost 231 orders surveyed, there are two lhfpl5 genes whose protein products cluster with either the lhfpl5a or 232 lhfpl5b ohnolog groups. Based on available genomic data, there is no evidence that spotted gar fish 233 have duplicated lhfpl5 genes. Nor is there evidence that the Salmonid-specific WGD (Allendorf and 234 Thorgaard, 1984) lead to further expansion of the lhfpl5 family. These analyses suggest that the 235 ancestral lhfpl5 gene was duplicated in the teleost WGD and that the lhfpl5 ohnologs were retained in 236 all teleost species examined here. 237 238 Alignment of the zebrafish Lhfpl5a and Lhfpl5b proteins with those from human, mouse and chicken 239 reveals that both zebrafish ohnologs retain the same protein structure ( Figure 1B) To characterize the spatial and temporal patterns of lhfpl5a/b gene expression, we performed whole 249 mount mRNA in situ hybridization on zebrafish larvae at 1, 2, and 5-days post-fertilization (dpf) 250 ( Figure 2). After one day of development, there are nascent hair cells of the presumptive anterior and 251 posterior maculae in the developing ear, but no lateral line hair cells at this stage. We detect lhfpl5a 252 expression in the presumptive anterior and posterior maculae at 1 dpf ( Figure 2A). No signal for lhfpl5b 253 was observed at this time point ( Figure 2B). At 2 dpf, we observe a clear distinction in the expression 254 patterns of the lhfpl5a and lhfpl5b genes ( Figure 2C, D). lhfpl5a continues to be expressed in the ear, 255 but expression is not observed in the newly deposited neuromasts. Conversely, we detect lhfpl5b 256 expression exclusively in neuromasts at this stage, both on the head ( Figure 2D) and trunk (data not 257 shown). This divergence in lhfpl5 ohnolog expression continues at 5 dpf, with lhfpl5a found 258 exclusively in the sensory patches of the ear and lhfpl5b restricted to lateral line hair cells ( Figure 2E-259 J). Taken together, our results suggest that both lhfpl5a and lhfpl5b have been retained since the teleost 260 whole genome duplication because of their non-overlapping mRNA expression patterns. 261

lhfpl5a mediates mechanosensitivity of otic hair cells. 262
lhfpl5a tm290d (astronaut / asn) mutants were initially characterized by balance defects, an absence of 263 the acoustic startle reflex, and a lack of brainstem Ca 2+ signals after acoustic startle. However, 264 neuromast microphonic potentials were normal (Nicolson et al., 1998). In light of our in situ 265 hybridization data (Figure 2), these results suggest that lhfpl5a is required for auditory and vestibular 266 hair cell function only. To test this possibility, we performed several functional assays measuring the 267 activity of hair cells in the otic capsule. First, we tested macular hair cell activity (Lu and DeSmidt,268 2013; Yao et al., 2016) by recording extracellular microphonic potentials from the inner ears of 269 lhfpl5a tm290d mutants, along with wild type and lhfpl5b vo35 larvae at 3 dpf. We then measured baseline-270 to-peak amplitude of the first peak to quantify activity (Supplemental Figure 1A-D). Consistent with 271 the initial characterization of the behavioral defects in lhfpl5a tm290d mutants, we did not detect robust 272 microphonic potentials from the inner ear of lhfpl5a tm290d mutants ( Figure 3A). We measured an 273 average first peak microphonic of 53.1 µV from lhfpl5a tm290d mutants (n = 7), which was significantly 274 less than the 296.2 µV average value of their WT siblings (n = 5; p = 0.01, Welch's t-test). We believe 275 that at least some of the signal detected in lhfpl5a tm290d mutants may be a stimulus artefact due the 276 unusual rise time and similar observations we have made in other known transduction-null mutants. 277 For the lhfpl5b vo35 mutants (n = 6), we measured an average first peak microphonic potential of 280.9 278 µV. These values were not significantly different from their WT siblings (n = 4, average 243.8 µV; p 279 = 0.7) but were significantly greater than the lhfpl5a tm290d mutants (p = 0.036). Thus, lhfpl5a tm290d 280 mutants exhibit defects in inner ear function, while the lhfpl5b vo35 mutation has no effect on these hair 281 cells. To further confirm the lack of inner ear function in lhfpl5a tm290d mutants, we performed an 282 acoustic startle test on larvae at 6 dpf ( Figure 3B). Results confirm that lhfpl5a tm290d mutants are 283 profoundly deaf, displaying little to no response to acoustic stimuli (p < 0.001 compared to all other 284 genotypes). This is a provisional file, not the final typeset article demonstrate that the GFP-Lhfpl5a protein is functional, we assayed for rescue of the acoustic startle 293 reflex and MET channel activity in homozygous lhfpl5a tm290d mutants expressing the transgene. The 294 startle reflex of lhfpl5a tm290d mutants expressing GFP-Lhfpl5a were statistically indistinguishable from 295 non-transgenic and transgenic WT siblings (p = 0.48 and 0.26 respectively; Figure 3B). Likewise, FM 296 4-64 dye-labeling in the inner ear was restored to lhfpl5a tm290d mutants expressing GFP-Lhfpl5a ( Figure  297 3E-F'). From these results we conclude that the hair bundle-localized GFP-Lhfpl5a protein is 298 functional and can rescue the behavioral and MET channel defects in lhfpl5a tm290d mutants. cells. To test this idea, we compared hair cell counts between wild type, lhfpl5a tm290d , and lhfpl5b vo35 321 larvae at 5 dpf. As expected, wild type and lhfpl5a tm290d mutants have statistically equivalent numbers 322 of hair cells in the neuromasts surveyed (Supplemental Figure 2D, p = 0.58). In lhfpl5b vo35 mutant 323 neuromasts, we observe a statistically significant decrease in the number of hair cells ( Figure 4K, 324 n = 11 individuals per genotype, 3 NM each, p < 0.001 each NM type by Welch's t-test; representative 325 neuromasts are shown in Supplemental Figure 4). This decrease in the number of neuromast hair cells 326 is consistent with lhfpl5b mutants being deficient in mechanotransduction in this cell type. 327

lhfpl5 is not required for Tmc localization to the hair bundle in zebrafish hair cells. 328
Previous studies have demonstrated that loss of LHFPL5 leads to a ~90% reduction in the peak 329 amplitude of the transduction current in mouse cochlear outer hair cells ( GFP and Tmc2b-GFP are still targeted to the hair bundle in lhfpl5a tm290d mutants (n = 11/11 and 9/9 341 individuals respectively; Figure 5A-D'). We also observe Tmc2b-GFP localization in the neuromast 342 hair bundles of lhfpl5b vo35 mutants (n = 7/7 individuals; Figure 5E-F'). Additionally, we do not observe 343 any rescue of hair cell function in lhfpl5b mutants expressing the Tmc2b-GFP protein (Supplemental 344 Figure 5A-C). As such, these results suggest that GFP-tagged Tmc1 and Tmc2b do not require Lhfpl5 345 for hair bundle localization in zebrafish hair cells. part of the hair bundle, with less intense punctate staining throughout the stereocilia in 3 dpf wild type 354 larvae ( Figure 6A). lhfpl5a tm290d mutants exhibit splayed hair bundles, with low levels of Pcdh15a 355 staining restricted to the tip of each stereocilium (n = 5/5 individuals; Figure 6B), confirming our 356 previous report. 357 To determine if Lhfpl5a also depends on Pcdh15a for its localization, we imaged GFP-Lhfpl5 in the 358 lateral cristae of pcdh15a psi7 homozygous mutants. In wild type larvae, GFP-Lhfpl5a is distributed 359 throughout the hair bundle at the tips of stereocilia (yellow arrow heads), with a higher intensity of 360 GFP evident in the tallest rows, possibly at the sites of kinociliary links ( Figure 6C, C', arrow). In 361 pcdh15a psi7 mutants, GFP-Lhfpl5a is absent from the hair bundle except for the tallest rows of 362 stereocilia adjacent to the kinocilium (n = 4 individuals; Figure 6D, D', arrow). We observed similar 363 results using the pcdh15a th263b allele (n = 6/6 individuals; Supplemental Figure 6A, B whether Cdh23 was required for GFP-Lhfpl5a localization. In cdh23 nl9 mutants, GFP-Lhfpl5 is 374 sparsely distributed throughout the length of the kinocilium (n = 4/4 individuals, Figure 6E, E'). We 375 also observe a redistribution of GFP-Lhfpl5a into the kinocilium using the cdh23 tj264 allele (n = 4/4 376 individuals; Supplemental Figure 6C). In both cdh23 alleles, we see occasional GFP signal in the 377 stereocilia as well, though this localization is not as robust. its role in Lhfpl5 localization has not been examined. In myo7aa ty220 (mariner) mutant hair cells, GFP-389 Lhfpl5a localization in hair bundle is severely disrupted (n = 5/5 individuals; Figure 6 F, F'). We 390 observe GFP signal near the base of hair bundles and occasionally in stereocilia (yellow arrowheads), 391 but do not see robust Lhfpl5a localization in the presumptive kinocilial links nor to the kinocilium 392 itself. Taken together, our results using the GFP-Lhfpl5a transgene suggest that Pcdh15a, Cdh23, and 393 Myo7aa all play distinct roles in Lhfpl5 localization in the bundle of zebrafish vestibular hair cells. 394

5
Discussion 395 In this study, we provide support for the following points: 1) The ancestral lhfpl5 gene was likely 396 duplicated as a result of the teleost whole genome duplication, leading to the lhfpl5a and lhfpl5b 397 ohnologs described in this paper. 2) In zebrafish, there has been subfunctionalization of these genes as and functionally required in distinct, non-overlapping hair cell lineages. To our knowledge, this is the 415 first description of duplicated genes whose expression patterns have cleanly partitioned between inner 416 ear and lateral line hair cells. 417

Subfunctionalization of zebrafish lhfpl5 ohnologs. 418
We constructed a phylogenetic tree using publicly available Lhfpl5 protein sequence data (Figure 1) 419 Our results suggest that the ancestral lhfpl5 gene was duplicated during the teleost WGD event and that 420 both genes have been retained throughout the teleost lineage (Figure 1). Our conclusion that lhfpl5a 421 and lhfpl5b are ohnologs is substantiated by a recent study of ohnologs in teleosts (Singh and Isambert,422 2019). In all four teleost species surveyed, Singh and Isambert show that lhfpl5a and lhfpl5b are true 423 ohnologs that arose from the teleost-specific WGD under the strictest criteria used in their study. 424 Some kind of selection pressure is required for the retention of both gene ohnologs (Glasauer and 425 Neuhauss, 2014). Our in situ hybridization results show that lhfpl5a and lhfpl5b are expressed in 426 distinct populations of hair cells and support the idea that the zebrafish lhfpl5 ohnologs were retained 427 because of their divergent expression patterns (Figure 2). Analysis of hair cell function in the different 428 lhfpl5 mutants agrees with the gene expression results, showing that lhfpl5a is required for transduction 429 in the ear while lhfpl5b plays the same role in the lateral line organs. However, rescue of the MET 430 channel defects in lhfpl5b vo35 mutants by GFP-tagged Lhfpl5a suggests that the functions of these 431 ohnologs are at least partially interchangeable. This result is not surprising given the high degree of 432 similarity between the Lhfpl5a and 5b proteins. As such, the subfunctionalization of the lhfpl5 ohnologs 433 appears to be caused by the divergence in their expression patterns rather than functional differences 434 in their protein products. It is possible that a similar mechanism is responsible for the retention of both 435 genes in other teleost species as well, though the expression patterns of the lhfpl5 ohnologs have not 436 been examined in other fish. 437 The non-overlapping expression of the lhfpl5 ohnologs provides us with a unique genetic tool to study 438 lateral line function. Most mechanotransduction mutants in zebrafish disrupt both inner ear and lateral 439 line hair cell function (Nicolson, 2017). As such, we are unable to assess the role of the lateral line 440 independently of auditory and vestibular defects, which are lethal for larval fish. The lhfpl5b mutants 441 are adult viable (data not shown) and represent a possible genetic model for understanding the lateral 442 line in both larval and adult fish. Future studies on adults will determine whether the lateral line remains 443 non-functional and whether lhfpl5b contributes to inner ear function in the mature auditory and 444 vestibular systems. 445

The molecular requirements for bundle localization of MET complex proteins differs 446
between mouse and zebrafish hair cells.  to examine Lhfpl5 localization. In wild type hair cells, GFP-Lhfpl5a is present at the tips of the shorter 497 ranked stereocilia, but the most robust signal is detected in the tallest stereocilia adjacent to the 498 kinocilium. In pcdh15a mutants, only this "kinocilial link"-like signal remains in the hair bundle. This 499 result is similar to the LHFPL5 localization reported at the tips of the tallest stereocilia of P3 cochlear 500 hair cells from wild type mice, a region where LHFPL5 would presumably not be associated with either 501 PCDH15 or the TMCs (Mahendrasingam et al., 2017). Since the kinocilium does not degenerate in 502 zebrafish hair cells, our results suggest that Lhfpl5a is normally targeted to these kinocilial links in 503 wild type cells and that its retention at this site does not require Pcdh15a. Thus, there are PCDH15-504 dependent and PCDH15-independent mechanisms of LHFPL5 localization in both mouse and 505 zebrafish hair cells. These results suggest that LHFPL5 performs as-of-yet uncharacterized PCDH15-506 independent functions as well. 507 Cdh23 is thought to form part of the linkages between the kinocilium and adjacent stereocilia, and 508 therefore may play a role in retaining Lhfpl5a at these sites. Consistent with this hypothesis, cdh23 509 mutants do not exhibit GFP-Lhfpl5a accumulation in stereocilia. Instead, GFP-Lhfpl5a is diffusely 510 distributed throughout the kinocilium. These data suggest that normal Lhfpl5a localization requires 511 both Pcdh15a and Cdh23 function, albeit in distinct ways. This result differs from previous reports 512 which found that Lhfpl5 still localized to the tips of stereocilia in CDH23-deficient cochlear hair cells 513 of mice. The same study also could not detect a biochemical interaction between Lhfpl5 and CDH23 514 via co-IP in heterologous cells (Xiong et al., 2012). Given these contrasting results for mouse and 515 zebrafish hair cells, it seems that the requirement for Cdh23 in the localization of LHFPL5 is not a 516 universal one. 517 Given the available biochemical evidence, it is possible that the association between Lhfpl5a and 518 Cdh23 is indirect through an as-of-yet unidentified protein or protein complex. Based on the defects in 519 GFP-Lhfpl5a localization in myo7aa mutants, Myo7aa is a candidate member of this uncharacterized 520 protein complex. Taken together, these results highlight the fact that, although all sensory hair cells 521 share many core genetic and biochemical features, there are important details that differ between the 522 various types of hair cells and between vertebrate species. Analyzing these differences will allow for 523 a more comprehensive understanding of vertebrate hair cell function and the underlying principles of 524 mechanotransduction. 525

6
Conflict of Interest 526 The authors declare that the research was conducted in the absence of any commercial or financial 527 relationships that could be construed as a potential conflict of interest. 528   Table 1. Lhfpl5 proteins used to construct the phylogenetic tree in Figure 1A.