Improved Locomotor Recovery in a Rat Model of Spinal Cord Injury by BioLuminescent-OptoGenetic (BL-OG) Stimulation with an Enhanced Luminopsin.

Irrespective of the many strategies focused on dealing with spinal cord injury (SCI), there is still no way to restore motor function efficiently or an adequate regenerative therapy. One promising method that could potentially prove highly beneficial for rehabilitation in patients is to re-engage specific neuronal populations of the spinal cord following SCI. Targeted activation may maintain and strengthen existing neuronal connections and/or facilitate the reorganization and development of new connections. BioLuminescent-OptoGenetics (BL-OG) presents an avenue to non-invasively and specifically stimulate neurons; genetically targeted neurons express luminopsins (LMOs), light-emitting luciferases tethered to light-sensitive channelrhodopsins that are activated by adding the luciferase substrate coelenterazine (CTZ). This approach employs ion channels for current conduction while activating the channels through treatment with the small molecule CTZ, thus allowing non-invasive stimulation of all targeted neurons. We previously showed the efficacy of this approach for improving locomotor recovery following severe spinal cord contusion injury in rats expressing the excitatory luminopsin 3 (LMO3) under control of a pan-neuronal and motor-neuron-specific promoter with CTZ applied through a lateral ventricle cannula. The goal of the present study was to test a new generation of LMOs based on opsins with higher light sensitivity which will allow for peripheral delivery of the CTZ. In this construct, the slow-burn Gaussia luciferase variant (sbGLuc) is fused to the opsin CheRiff, creating LMO3.2. Taking advantage of the high light sensitivity of this opsin, we stimulated transduced lumbar neurons after thoracic SCI by intraperitoneal application of CTZ, allowing for a less invasive treatment. The efficacy of this non-invasive BioLuminescent-OptoGenetic approach was confirmed by improved locomotor function. This study demonstrates that peripheral delivery of the luciferin CTZ can be used to activate LMOs expressed in spinal cord neurons that employ an opsin with increased light sensitivity.

Defining parameters of specificity for bioluminescent optogenetic activation of neurons using in vitro multi electrode arrays (MEA).

In Bioluminescent Optogenetics (BL-OG) a biological, rather than a physical, light source is used to activate light-sensing opsins, such as channelrhodopsins or pumps. This is commonly achieved by utilizing a luminopsin (LMO), a fusion protein of a light-emitting luciferase tethered to a light-sensing opsin. Light of the wavelength matching the activation peak of the opsin is emitted by the luciferase upon application of its small molecule luciferin, resulting in activation of the fused opsin and subsequent effects on membrane potential. Using optimized protocols for culturing, transforming, and testing primary neurons in multi electrode arrays, we systematically defined parameters under which changes in neuronal activity are specific to bioluminescent activation of opsins, rather than due to off-target effects of either the luciferin or its solvent on neurons directly, or on opsins directly. We further tested if there is a direct effect of bioluminescence on neurons. Critical for assuring specific BL-OG effects are testing the concentration and formulation of the luciferin against proper controls, including testing effects of vehicle on LMO expressing and of luciferin on nonLMO expressing targets.

Bioluminescence-driven optogenetic activation of transplanted neural precursor cells improves motor deficits in a Parkinson's disease mouse model.

The need to develop efficient therapies for neurodegenerative diseases is urgent, especially given the increasing percentages of the population living longer, with increasing chances of being afflicted with conditions like Parkinson's disease (PD). A promising curative approach toward PD and other neurodegenerative diseases is the transplantation of stem cells to halt and potentially reverse neuronal degeneration. However, stem cell therapy does not consistently lead to improvement for patients. Using remote stimulation to optogenetically activate transplanted cells, we attempted to improve behavioral outcomes of stem cell transplantation. We generated a neuronal precursor cell line expressing luminopsin 3 (LMO3), a luciferase-channelrhodopsin fusion protein, which responds to the luciferase substrate coelenterazine (CTZ) with emission of blue light that in turn activates the opsin. Neuronal precursor cells were injected bilaterally into the striatum of homozygous aphakia mice, which carry a spontaneous mutation leading to lack of dopaminergic neurons and symptoms of PD. Following transplantation, the cells were stimulated over a period of 10 days by intraventricular injections of CTZ. Mice receiving CTZ demonstrated significantly improved motor skills in a rotarod test compared to mice receiving vehicle. Thus, bioluminescent optogenetic stimulation of transplanted neuronal precursor cells shows promising effects in improving locomotor behavior in the aphakia PD mouse model and encourages further studies to elucidate the mechanisms and long-term outcomes of these beneficial effects.

Engineering luminopsins with improved coupling efficiencies.

Significance: Luminopsins (LMOs) are bioluminescent-optogenetic tools with a luciferase fused to an opsin that allow bimodal control of neurons by providing both optogenetic and chemogenetic access. Determining which design features contribute to the efficacy of LMOs will be beneficial for further improving LMOs for use in research. Aim: We investigated the relative impact of luciferase brightness, opsin sensitivity, pairing of emission and absorption wavelength, and arrangement of moieties on the function of LMOs. Approach: We quantified efficacy of LMOs through whole cell patch clamp recordings in HEK293 cells by determining coupling efficiency, the percentage of maximum LED induced photocurrent achieved with bioluminescent activation of an opsin. We confirmed key results by multielectrode array recordings in primary neurons. Results: Luciferase brightness and opsin sensitivity had the most impact on the efficacy of LMOs, and N-terminal fusions of luciferases to opsins performed better than C-terminal and multi-terminal fusions. Precise paring of luciferase emission and opsin absorption spectra appeared to be less critical. Conclusions: Whole cell patch clamp recordings allowed us to quantify the impact of different characteristics of LMOs on their function. Our results suggest that coupling brighter bioluminescent sources to more sensitive opsins will improve LMO function. As bioluminescent activation of opsins is most likely based on Förster resonance energy transfer, the most effective strategy for improving LMOs further will be molecular evolution of luciferase-fluorescent protein-opsin fusions.

Efficient opto- and chemogenetic control in a single molecule driven by FRET-modified bioluminescence.

Significance: Bioluminescent optogenetics (BL-OG) offers a unique and powerful approach to manipulate neural activity both opto- and chemogenetically using a single actuator molecule (a LuMinOpsin, LMO). Aim: To further enhance the utility of BL-OG by improving the efficacy of chemogenetic (bioluminescence-driven) LMO activation. Approach: We developed novel luciferases optimized for Förster resonance energy transfer when fused to the fluorescent protein mNeonGreen, generating bright bioluminescent (BL) emitters spectrally tuned to Volvox Channelrhodopsin 1 (VChR1). Results: A new LMO generated from this approach (LMO7) showed significantly stronger BL-driven opsin activation compared to previous and other new variants. We extensively benchmarked LMO7 against LMO3 (current standard) and found significantly stronger neuronal activity modulation ex vivo and in vivo, and efficient modulation of behavior. Conclusions: We report a robust new option for achieving multiple modes of control in a single actuator and a promising engineering strategy for continued improvement of BL-OG.

A New Highly Efficient Molecule for Both Optogenetic and Chemogenetic Control Driven by FRET Amplification of BioLuminescence.

SIGNIFICANCE: Bioluminescent optogenetics (BL-OG) offers a unique and powerful approach to manipulate neural activity both opto- and chemogenetically using a single actuator molecule (a LuMinOpsin, LMO). AIM: To further enhance the utility of BL-OG by improving the efficacy of chemogenetic (bioluminescence-driven) LMO activation. APPROACH: We developed novel luciferases optimized for Forster resonance energy transfer (FRET) when fused to the fluorescent protein mNeonGreen, generating bright bioluminescent (BL) emitters spectrally tuned to Volvox Channelrhodopsin 1 (VChR1). RESULTS: A new LMO generated from this approach (LMO7) showed significantly stronger BL-driven opsin activation compared to previous and other new variants. We extensively benchmarked LMO7 against LMO3 (current standard), and found significantly stronger neuronal activity modulation ex vivo and in vivo, and efficient modulation of behavior. CONCLUSIONS: We report a robust new option for achieving multiple modes of control in a single actuator, and a promising engineering strategy for continued improvement of BL-OG.

A Bioluminescent Activity Dependent (BLADe) Platform for Converting Neuronal Activity to Photoreceptor Activation.

We developed a platform that utilizes a calcium-dependent luciferase to convert neuronal activity into activation of light sensing domains within the same cell. The platform is based on a Gaussia luciferase variant with high light emission split by calmodulin-M13 sequences that depends on influx of calcium ions (Ca2+) for functional reconstitution. In the presence of its luciferin, coelenterazine (CTZ), Ca2+ influx results in light emission that drives activation of photoreceptors, including optogenetic channels and LOV domains. Critical features of the converter luciferase are light emission low enough to not activate photoreceptors under baseline condition and high enough to activate photosensing elements in the presence of Ca2+ and luciferin. We demonstrate performance of this activity-dependent sensor and integrator for changing membrane potential and driving transcription in individual and populations of neurons in vitro and in vivo.

Engineering luminopsins with improved coupling efficiencies.

Significance: Luminopsins (LMOs) are bioluminescent-optogenetic tools with a luciferase fused to an opsin that allow bimodal control of neurons by providing both optogenetic and chemogenetic access. Determining which design features contribute to the efficacy of LMOs will be beneficial for further improving LMOs for use in research. Aim: We investigated the relative impact of luciferase brightness, opsin sensitivity, pairing of emission and absorption wavelength, and arrangement of moieties on the function of LMOs. Approach: We quantified efficacy of LMOs through whole cell patch clamp recordings in HEK293 cells by determining coupling efficiency, the percentage of maximum LED induced photocurrent achieved with bioluminescent activation of an opsin. We confirmed key results by multielectrode array (MEAs) recordings in primary neurons. Results: Luciferase brightness and opsin sensitivity had the most impact on the efficacy of LMOs, and N-terminal fusions of luciferases to opsins performed better than C-terminal and multi-terminal fusions. Precise paring of luciferase emission and opsin absorption spectra appeared to be less critical. Conclusions: Whole cell patch clamp recordings allowed us to quantify the impact of different characteristics of LMOs on their function. Our results suggest that coupling brighter bioluminescent sources to more sensitive opsins will improve LMO function. As bioluminescent activation of opsins is most likely based on Förster resonance energy transfer (FRET), the most effective strategy for improving LMOs further will be molecular evolution of luciferase-fluorescent protein-opsin fusions.

Selective control of synaptically-connected circuit elements by all-optical synapses.

Understanding percepts, engrams and actions requires methods for selectively modulating synaptic communication between specific subsets of interconnected cells. Here, we develop an approach to control synaptically connected elements using bioluminescent light: Luciferase-generated light, originating from a presynaptic axon terminal, modulates an opsin in its postsynaptic target. Vesicular-localized luciferase is released into the synaptic cleft in response to presynaptic activity, creating a real-time Optical Synapse. Light production is under experimenter-control by introduction of the small molecule luciferin. Signal transmission across this optical synapse is temporally defined by the presence of both the luciferin and presynaptic activity. We validate synaptic Interluminescence by multi-electrode recording in cultured neurons and in mice in vivo. Interluminescence represents a powerful approach to achieve synapse-specific and activity-dependent circuit control in vivo.

Bioluminescent Optogenetics 2.0: Harnessing Bioluminescence to Activate Photosensory Proteins In Vitro and In Vivo.

Bioluminescence - light emitted by a luciferase enzyme oxidizing a small molecule substrate, a luciferin - has been used in vitro and in vivo to activate light-gated ion channels and pumps in neurons. While this bioluminescent optogenetics (BL-OG) approach confers a chemogenetic component to optogenetic tools, it is not limited to use in neuroscience. Rather, bioluminescence can be harnessed to activate any photosensory protein, thus enabling the manipulation of a multitude of light-mediated functions in cells. A variety of luciferase-luciferin pairs can be matched with photosensory proteins requiring different wavelengths of light and light intensities. Depending on the specific application, efficient light delivery can be achieved by using luciferase-photoreceptor fusion proteins or by simple co-transfection. Photosensory proteins based on light-dependent dimerization or conformational changes can be driven by bioluminescence to effect cellular processes from protein localization, regulation of intracellular signaling pathways to transcription. The protocol below details the experimental execution of bioluminescence activation in cells and organisms and describes the results using bioluminescence-driven recombinases and transcription factors. The protocol provides investigators with the basic procedures for carrying out bioluminescent optogenetics in vitro and in vivo. The described approaches can be further extended and individualized to a multitude of different experimental paradigms.

Bioluminescent optogenetic (BL-OG) activation of neurons during mouse postnatal brain development.

Bioluminescent optogenetics (BL-OG) allows activation of photosensory proteins, such as opsins, by either fiberoptics or by administering a luciferin. BL-OG thus confers both optogenetic and chemogenetic access within the same genetically targeted neuron. This bimodality offers a powerful approach for non-invasive chemogenetic manipulation of neural activity during brain development and adult behaviors with standard optogenetic spatiotemporal precision. We detail protocols for bioluminescent stimulation of neurons in postnatally developing brain and its validation through bioluminescence imaging and electrophysiological recording in mice. For complete information on the use and execution of this protocol, please refer to Medendorp et al. (2021).

Selective postnatal excitation of neocortical pyramidal neurons results in distinctive behavioral and circuit deficits in adulthood.

In genetic and pharmacological models of neurodevelopmental disorders, and human data, neural activity is altered within the developing neocortical network. This commonality begs the question of whether early enhancement in excitation might be a common driver, across etiologies, of characteristic behaviors. We tested this concept by chemogenetically driving cortical pyramidal neurons during postnatal days 4-14. Hyperexcitation of Emx1-, but not dopamine transporter-, parvalbumin-, or Dlx5/6-expressing neurons, led to decreased social interaction and increased grooming activity in adult animals. In vivo optogenetic interrogation in adults revealed decreased baseline but increased stimulus-evoked firing rates of pyramidal neurons and impaired recruitment of inhibitory neurons. Slice recordings in adults from prefrontal cortex layer 5 pyramidal neurons revealed decreased intrinsic excitability and increased synaptic E/I ratio. Together these results support the prediction that enhanced pyramidal firing during development, in otherwise normal cortex, can selectively drive altered adult circuit function and maladaptive changes in behavior.

Restoring Function After Severe Spinal Cord Injury Through BioLuminescent-OptoGenetics.

The ability to manipulate specific neuronal populations of the spinal cord following spinal cord injury (SCI) could prove highly beneficial for rehabilitation in patients through maintaining and strengthening still existing neuronal connections and/or facilitating the formation of new connections. A non-invasive and highly specific approach to neuronal stimulation is bioluminescent-optogenetics (BL-OG), where genetically expressed light emitting luciferases are tethered to light sensitive channelrhodopsins (luminopsins, LMO); neurons are activated by the addition of the luciferase substrate coelenterazine (CTZ). This approach utilizes ion channels for current conduction while activating the channels through the application of a small chemical compound, thus allowing non-invasive stimulation and recruitment of all targeted neurons. Rats were transduced in the lumbar spinal cord with AAV2/9 to express the excitatory LMO3 under control of a pan-neuronal or motor neuron-specific promoter. A day after contusion injury of the thoracic spine, rats received either CTZ or vehicle every other day for 2 weeks. Activation of either neuron population below the level of injury significantly improved locomotor recovery lasting beyond the treatment window. Utilizing histological and gene expression methods we identified neuronal plasticity as a likely mechanism underlying the functional recovery. These findings provide a foundation for a rational approach to spinal cord injury rehabilitation, thereby advancing approaches for functional recovery after SCI. SUMMARY: Bioluminescent optogenetic activation of spinal neurons results in accelerated and enhanced locomotor recovery after spinal cord injury in rats.

Diverse pathological implications of YKL-40: answers may lie in 'outside-in' signaling.

The developing paradigms about YKL-40, a member of the "mammalian chitinase-like proteins", from across the globe, project it as a vital parameter for the detection of disease onset and progression. It is expressed and secreted by cancer cells of different origins along with a variety of non-malignant cells including inflammatory and structural cells. Numerous studies demonstrate that YKL-40 over-expression is associated with increased patient mortality though the cellular receptors responsible for mediating these effects have not yet been identified. The putative YKL-40 ligands are thought to be carbohydrate structures, since it is capable of binding chitin, chito-oligosaccharides and heparin. Binding of collagen to YKL-40, identified it as the only non-carbohydrate extracellular matrix (ECM) ligand for YKL-40. Our broad understanding of YKL-40 as a versatile biomarker and its involvement in activating several signaling pathways make us anticipate that its specific receptors/binding partners may exist on the cell surface also. The cell surface heparan sulfate (HS) moieties seem to be the potential candidates for this role, suggesting that it could interact with HS-proteoglycans. It is recommended to clearly delineate YKL-40-mediated signaling mechanisms before promoting the YKL-40 know-how for translational research, in both diagnostic and therapeutic applications. The present review provides an overview of YKL-40 as a versatile biomarker, discussing the related pathological mechanisms and aims to reassess and unify the already proposed diverse hypotheses in YKL-40-regulated signaling mechanisms.

Actinomycetes: a repertory of green catalysts with a potential revenue resource.

Biocatalysis, one of the oldest technologies, is becoming a favorable alternative to chemical processes and a vital part of green technology. It is an important revenue generating industry due to a global market projected at $7 billion in 2013 with a growth of 6.7% for enzymes alone. Some microbes are important sources of enzymes and are preferred over sources of plant and animal origin. As a result, more than 50% of the industrial enzymes are obtained from bacteria. The constant search for novel enzymes with robust characteristics has led to improvisations in the industrial processes, which is the key for profit growth. Actinomycetes constitute a significant component of the microbial population in most soils and can produce extracellular enzymes which can decompose various materials. Their enzymes are more attractive than enzymes from other sources because of their high stability and unusual substrate specificity. Actinomycetes found in extreme habitats produce novel enzymes with huge commercial potential. This review attempts to highlight the global importance of enzymes and extends to signify actinomycetes as promising harbingers of green technology.

53BP1 is limiting for NHEJ repair in ATM-deficient model systems that are subjected to oncogenic stress or radiation.

UNLABELLED: The DNA damage response (DDR) factors ataxia telangiectasia mutated (ATM) and p53 binding protein 1 (53BP1) function as tumor suppressors in humans and mice, but the significance of their mutual interaction to the suppression of oncogenic translocations in vivo has not been investigated. To address this question, the phenotypes of compound mutant mice lacking 53BP1 and ATM (Trp53bp1(-/-)/Atm(-/-)), relative to single mutants, were examined. These analyses revealed that loss of 53BP1 markedly decreased the latency of T-lineage lymphomas driven by RAG-dependent oncogenic translocations in Atm(-/-) mice (average survival, 14 and 23 weeks for Trp53bp1(-/-)/Atm(-/-) and Atm(-/-) mice, respectively). Mechanistically, 53BP1 deficiency aggravated the deleterious effect of ATM deficiency on nonhomologous end-joining (NHEJ)-mediated double-strand break repair. Analysis of V(D)J recombinase-mediated coding joints and signal joints in Trp53bp1(-/-)/Atm(-/-) primary thymocytes is, however, consistent with canonical NHEJ-mediated repair. Together, these findings indicate that the greater NHEJ defect in the double mutant mice resulted from decreased efficiency of rejoining rather than switching to an alternative NHEJ-mediated repair mechanism. Complementary analyses of irradiated primary cells indicated that defects in cell-cycle checkpoints subsequently function to amplify the NHEJ defect, resulting in more frequent chromosomal breaks and translocations in double mutant cells throughout the cell cycle. Finally, it was determined that 53BP1 is dispensable for the formation of RAG-mediated hybrid joints in Atm(-/-) thymocytes but is required to suppress large deletions in a subset of hybrid joints. IMPLICATIONS: The current study uncovers novel ATM-independent functions for 53BP1 in the suppression of oncogenic translocations and in radioprotection.

Hyaluronan-binding protein 1 (HABP1/p32/gC1qR) induces melanoma cell migration and tumor growth by NF-kappa B dependent MMP-2 activation through integrin α(v)ß(3) interaction.

Cell migration is the hallmark of cancer regulating anchorage independent growth and invasiveness of tumor cells. Hyaluronan (HA), an ECM polysaccharide is shown to regulate this process. In the present report, we demonstrated, supplementation of purified recombinant hyaluronan binding protein 1(HABP1/p32/gC1qR) from human fibroblast cDNA enhanced migration potential of highly invasive melanoma (B16F10) cells. Exogenous HABP1 adhered to the cell surface transiently and was shown to interact and colocalize with α(v)ß(3) integrin, a regulatory molecule of cell migration. In HABP1 treated cells, the phosphorylation of nuclear factor inducing kinase (NIK) and IκBα was observed, followed by nuclear translocation of p65 subunit of NFκB, along with its DNA-binding and transactivation, resulting in upregulation of MT1-MMP expression and finally MMP-2 activation. To substantiate our findings, prior to HABP1 treatment, the expression of NIK was reduced by small interfering RNA mediated knockdown and confirmed the inhibition of nuclear translocation of p65 subunit of NFκB and upregulation of MT1-MMP expression. In addition, the use of curcumin, an anti-cancer drug, or GRGDSP, the blocking peptide along with exogenous HABP1, inhibited such NFκB-dependent pathway, confirming that HABP1-induced cell migration is α(v)ß(3) integrin-mediated and downstream signaling by NFκB. Finally, we translated the in vitro data in mice model and observed enhanced tumor growth with higher MT1-MMP expression and MMP-2 activation in the tumors upon injection of HABP1 treated melanoma cells. The treatment of curcumin, the anticancer drug along with HABP1, inhibited the migration, expression of MT1-MMP and activation of MMP-2 and finally tumor growth supports the involvement of HABP1 in tumor formation.

Differential requirement for H2AX and 53BP1 in organismal development and genome maintenance in the absence of poly(ADP)ribosyl polymerase 1.

Combined deficiencies of poly(ADP)ribosyl polymerase 1 (PARP1) and ataxia telangiectasia mutated (ATM) result in synthetic lethality and, in the mouse, early embryonic death. Here, we investigated the genetic requirements for this lethality via analysis of mice deficient for PARP1 and either of two ATM-regulated DNA damage response (DDR) factors: histone H2AX and 53BP1. We found that, like ATM, H2AX is essential for viability in a PARP1-deficient background. In contrast, deficiency for 53BP1 modestly exacerbates phenotypes of growth retardation, genomic instability, and organismal radiosensitivity observed in PARP1-deficient mice. To gain mechanistic insights into these different phenotypes, we examined roles for 53BP1 in the repair of replication-associated double-strand breaks (DSBs) in several cellular contexts. We show that 53BP1 is required for DNA-PKcs-dependent repair of hydroxyurea (HU)-induced DSBs but dispensable for RPA/RAD51-dependent DSB repair in the same setting. Moreover, repair of mitomycin C (MMC)-induced DSBs and sister chromatid exchanges (SCEs), two RAD51-dependent processes, are 53BP1 independent. Overall, our findings define 53BP1 as a main facilitator of nonhomologous end joining (NHEJ) during the S phase of the cell cycle, beyond highly specialized lymphocyte rearrangements. These findings have important implications for our understanding of the mechanisms whereby ATM-regulated DDR prevents human aging and cancer.