Near-Infrared Irradiation Affects Lipid Metabolism in Neuronal Cells

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Near-infrared irradiation affects lipid metabolism in neuronal cells, inducing lipid droplets formation Svitlana M. Levchenko, Andrey N Kuzmin, Tymish Y. Ohulchanskyy, Artem Pliss, Junle Qu, and Paras N. Prasad ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00508 • Publication Date (Web): 30 Nov 2018 Downloaded from http://pubs.acs.org on December 1, 2018

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ACS Chemical Neuroscience

Near-infrared irradiation affects lipid metabolism in neuronal cells, inducing lipid droplets formation

Svitlana M. Levchenko1, Andrey N. Kuzmin2,3, Tymish Y. Ohulchanskyy1*, Artem Pliss2,3, Junle Qu1*, Paras N. Prasad2* 1 Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong

Province, College of Optoelectronic Engineering, Shenzhen University, Shenzhen, Guangdong Province 518060, China 2

Institute for Lasers, Photonics and Biophotonics, University at Buffalo, State University of New York, Buffalo, NY 14260-3000, USA 3

Advanced Cytometry Instrumentation Systems, LLC, 640 Ellicott Street – Suite 499, Buffalo, NY, USA *Corresponding authors: [email protected], [email protected], [email protected] Abstract It is known that lipids play an outstanding role in cellular regulation and their dysfunction has been linked to many diseases. Thus, modulation of lipid metabolism may provide new pathways for disease treatment or prevention. In this work, near infrared (NIR) light was applied to modulate lipid metabolism, and increase intracellular lipid content in rat cortical neurons (RCN). Using label-free CARS microscopy, we have monitored the intracellular lipid content in RCN at a single cell level. A major increase in average level of lipid per cell after treatment with laser diode at 808 nm was found, nonlinearly dependent on the irradiation dose. Moreover, a striking formation of lipid droplets (LDs) in the irradiated RCN was discovered. Further experiments and analysis reveal a strong correlation between NIR light induced generation of reactive oxygen species (ROS), lipids level and LDs formation in RCN. Our findings can contribute to a development of therapeutic approaches for neurological disorders via NIR light control of lipid metabolism in neuronal cells. Keywords: NIR light, CARS imaging, ROS, lipids, neuronal cells. Introduction Lipids play an outstanding role in cellular metabolism and their dysfunction has been linked to many diseases 1-4. Within the past few years, a new intriguing connection between lipid metabolism and neurodegenerative disorders has been discovered by several groups

5-10.

In

particular, it was shown that disruption of homeostasis of lipid and glucose metabolism affects production and clearance of β-amyloid and tau phosphorylation, and induces neurodegeneration during the pathogenesis of Alzheimer’s disease (AD) 8. The interaction of neurotoxic proteins with 1 ACS Paragon Plus Environment

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certain lipids and modification of lipid composition in different cell compartments, in particular mitochondria, decisively impact cell death associated with neurodegeneration 9. It is well known that cellular lipids may exist in the form of lipid droplets (LDs) which are highly dynamic and multifunctional organelles involved in many cellular processes 11-13. LDs can significantly vary in size, from 0.01 μm to 5μm and even more, depending on cell type14, 15. Cytosolic LDs exist in a form of emulsion droplets or micellar like structure that comprise a lipid core surrounded by a phospholipid monolayer containing different proteins12,

16, 17.

The hydrophobic core of neutral

lipids in LDs stores metabolic energy and membrane components, making LDs hubs for lipid metabolism 11. A number of evidences appeared that besides the energy metabolism, LDs also participate in storage and turnover of hydrophobic proteins, vitamins, and other biomolecules, management of endoplasmic reticulum and protecting against some forms of cellular stress

13.

Furthermore, LDs formation correlates with intracellular level of reactive oxygen species (ROS), as it was demonstrated in cultured cells 5-7, 10. It is known that while the moderate levels of ROS contribute to the course of many cellular physiologic processes, most notably, cellular immunity, the maturation subcellular structures, cell division, induction of cellular signaling pathways, etc.

18,

an excess of ROS can result in cell

damage. Furthermore, an oxidative stress and related mitochondrial dysfunction are closely associated with different neurodegenerative disorders such as AD, Amyotrophic lateral sclerosis, Friedreich’s ataxia, Huntington’s disease, Multiple sclerosis, and Parkinson’s disease 19, 20. In the brain cells, the negative effect of ROS is often mitigated by the synthesis of LDs. For instance, in response to oxidative stress, the embryonic glia cells form LDs, which were found to limit the levels of ROS and protect both glia and neuroblasts from peroxidation chain reactions, damaging for many types of macromolecules 10. The LDs pool of the brain was shown to change dynamically during brain development, with LDs being particularly enriched in cortex glial cells 5. It was also reported by other groups that the mitochondrial dysfunction, which involves excessive ROS production, leads also to accumulation of LDs in glia cells 6. The authors further suggested that LDs accumulation is an evolutionarily conserved phenomenon coupled to mitochondrial dysfunction and represents an early indicator of a number of neurodegenerative diseases. It was also found that reducing the number and size of LDs accumulation in glia via lowering ROS, can significantly delay the onset of neurodegeneration 6. Just recently, the same group has discovered another conserved mechanism for neuron-glia metabolic cooperation. They showed that glial lactate can fuel neuronal lipogenesis in response to ROS; in turn, neuronal lipids are exported to glia cells and stored there as LDs 7. Considering the fact that LDs accumulation is one of the probable presymptomatic changes in neurodegenerative diseases, detection of LDs may serve as a 2 ACS Paragon Plus Environment

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promising hallmark for early diagnosis 6, while manipulation of lipid metabolism can provide the background for developing an effective therapeutic approach. In this regard, photobiomodulation (PBM), employing red or near-infrared (NIR) light to effect biological processes 21, may represent a promising therapeutic platform to relieve symptoms, slow down the progress and treat some brain disorders. The beneficial effects of PBM in a wide range of neurological disorders has been demonstrated numerous times, using both cell culture and animal models, and in some clinical trials 21-25. In particular, a number of publications showed that ~810nm laser irradiation of cultured neurons , rescues mitochondrial dysfunction after oxygenglucose deprivation 26, reverses many of the adverse consequences of neuronal excitotoxicity 27, reduces oxidative stress

24, 27

and decreases the elevated intracellular ROS levels in stressed

neurons, while increasing the ROS levels in normal unstressed neurons 27 etc. It was found that in normal cells, absorption of light by Cytochrome C oxidase (Cox) leads to an increase in the mitochondrial membrane potential (MMP) and a short burst of ROS production

24, 27, 28.

At the

same time, when MMP is low in response to pre-existing oxidative stress, PBM leads to an increase of MMP, and the production of ROS is reduced

24.

Taken together, these findings open the

possibility for stimulating or inhibiting the level of intracellular ROS by using NIR irradiation. Despite a number of evidences that demonstrate changes taking place at cellular level in response to irradiation with NIR light, the exact mechanism of PBM is still not till not fully clarified 24, 28, 29.

Stated boldly, a link between the ROS level and LDs formation has been already highlighted, as well as a relation between LDs in glia and neuronal cells and the development of neurodegenerative diseases. From the other standpoint, the ability of NIR to affect intracellular ROS level and provide beneficial effects in treatment of brain disorders was also demonstrated. However, the mechanisms of NIR actions are not known yet. To our knowledge, there is no data on the relation between LDs and NIR light. In this regard, a challenging and intriguing question arises whether lipid metabolism in neuronal cells can be modulated by NIR light. Aiming to verify this hypothesis, we implemented Coherent anti-Stokes Raman scattering (CARS) microscopy for monitoring in time, changes in the lipid content in live neuronal cells irradiated with different doses of NIR light. CARS is frequently used as a robust tool for label-free chemically selective imaging

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and quantitative analysis of lipids content in cells and tissues

38, 39.

This approach

unraveled an additional layer of information on the effect of NIR light on cellular processes, particularly lipid metabolism and LDs formation in rat cortical neuronal cells. Moreover, the obtained data provide an opportunity for optimizing light parameters, and developing a more effective approach to the treatment of neurodegenerative disorders. 3 ACS Paragon Plus Environment


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Results and Discussion In this study, we employed CARS microscopy for real-time monitoring and quantitative analysis of the NIR light effect on lipid metabolism and LDs formation in neuronal cells. Our results reveal a major enhancement in the CARS signal from lipids at 2840 cm−1, in response to the PBM treatment, as well as the formation and accumulation of LDs in the cytoplasm of RCN cells, as shown in Fig. 1(A, C, E) and Fig. S1(a, c) in Supporting Information. For quantitative representation of the lipid boost, 3-D surface plots of the lipid CARS signal intensities were generated and are presented aside the acquired CARS images (Fig. 1(B, D, F) and Fig. S1(b, d)). As one can see from Fig.1, the CARS signal of lipids in the cytoplasm drastically rises in 2 hours after the irradiation; along that, LDs formation is observed. Moreover, monitoring of the variations in the lipid content in time reveals that these changes are dose-dependent (Fig.1 and S1).

Figure 1 Real-time monitoring of the PBM effect on the lipids level in neuronal cells. Representative CARS images and corresponding intensity surface plots of RCN cells untreated in control (A and B), and RCN cells treated with PBM (0.3 and 30J/cm2, 808nm) before and 2h, 6h and 24h after irradiation (C-F). Surface plots were generated using ImageJ software. CARS images and surface plots for other tested doses (3 and 10 J/cm2) are presented in the Supporting information (Fig.S1). 4 ACS Paragon Plus Environment

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To verify our CARS observations and confirm the appearance of the LDs, we labeled neuronal cells with LDs-specific fluorescent probe, LipidTOX, which allowed us to detect lipid droplets with confocal fluorescence microscopy. It is worth noting that staining with LipidTOX™ requires cell fixation and, therefore, is not suitable for living cell studies. Confocal fluorescence microscopy images were acquired at the same time points before and after PBM treatment as CARS images, with identical doses of NIR irradiation. As expected, we found that a boost in the LDs formation can be also visualized with fluorescence imaging (Fig. S2, Supporting Information), confirming that PBM induces prominent changes in lipid metabolism and LDs formation in RCN. Next, we comparatively analyzed the lipid levels in the neuronal cell cytoplasm (determined from CARS signal intensity) in response to PBM, as described in Methods. Calculating lipid levels from CARS images of RCN 40, we found significant variations in the lipid content in RCN cells after treatment with different doses of laser irradiation (Fig. 2). The lipid content was analyzed as described in Materials and methods. For each tested dose, we observed a significant increase in the lipid level, 2h post irradiation (up to 7-8.5 times, if compared to the initial level) (Fig. 2A). Interestingly, 2h after the treatment with lower doses of 0.3 and 3J/cm2, the lipid level raised slightly less (~7.5 and ~7 times, correspondingly) than after treating with higher doses of 10 and 30 J/cm2 (~8.5 and ~8 times, correspondingly). However, at subsequent time points, the signal from lipids was found to increase more significantly in cells treated with a lowest dose of irradiation. In 6 and 24h after irradiation with 0.3J/cm2, the lipid level was ~17 and ~20 times higher than before treatment. In contrast, higher doses induce less significant changes in the cytoplasmic lipid content. RCN treatment with doses of 3, 10 and 30J/cm2 resulted in an increase in the lipid level up to 8-9 times and 11-13 times 6 and 24h after irradiation, respectively (Fig. 2A). Overall, our data indicate striking changes in the lipid content of neuronal cells after treatment with 808 nm light, nonlinearly dependent on the irradiation dose.

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Figure 2. (A) CARS signal acquired at the characteristic vibration of lipids in the cytoplasm of the RCN cells without (control) and with different doses of PBM (0.3, 3, 10 and 30 J/cm2, 808nm laser). (B) Quantification of LDs number per cell. The error bars correspond to standard deviations. Statistically significant differences from control are indicated as *(p

Near-Infrared Irradiation Affects Lipid Metabolism in Neuronal Cells

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