Tetramethylpyrazine nitrone protects retinal ganglion cells against N-methyl-d-aspartate-induced excitotoxicity.

Adding a free radical-scavenging nitrone moiety on tetramethylpyrazine, we have previously synthesized a chemical named 2-[[(1,1-dimethylethyl)oxidoimino]-methyl]-3,5,6-trimethylpyrazine (tetramethylpyrazine nitrone, or TBN) and proved its neuroprotective effect but with limited understanding of its mechanism. Here we ask if TBN protects retinal ganglion cells (RGCs) against excitotoxicity induced by NMDA and explore the underlying mechanism. NMDA was intravitreally injected to induce RGC injury in rats, followed by daily intraperitoneal administrations of TBN. Measurements of TBN concentration at different times after intraperitoneal administration showed that more than 200 µM TBN reached the aqueous humor quickly. Then RGCs' survival was evaluated by quantifying Brn3-positive cells, and retinal functions were examined by electroretinogram and visual behaviors. TBN significantly increased the survival of RGCs after NMDA insult, recovered the amplitude of photopic negative responses to flash, and restored the visual behavior. Furthermore, TBN inhibited the apoptotic process, as indicated by the elevated ratios of cleaved caspase-3/caspase-3 and of Bax/Bcl-2, and decreased the level of reactive oxygen species. Moreover, TBN reduced RGC's calcium overload induced by NMDA or by KCl. Whole-cell patch recording from RGCs further showed that TBN slightly but significantly inhibited L-type calcium channels, but had little effect on T-type calcium channel or NMDA-, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid(AMPA)-induced current. Thus our data indicate that TBN alleviates NMDA-elicited injury of rat RGCs both morphologically and functionally, possibly by inhibiting the L-type calcium channel thus reducing Ca2+ overload and by directly scavenging free radicals. Therefore, TBN may be a novel candidate for treating excitotoxicity-related visual disorders such as glaucoma.

Two-photon live imaging of direct glia-to-neuron conversion in the mouse cortex.

JOURNAL/nrgr/04.03/01300535-202408000-00032/figure1/v/2023-12-16T180322Z/r/image-tiff Over the past decade, a growing number of studies have reported transcription factor-based in situ reprogramming that can directly convert endogenous glial cells into functional neurons as an alternative approach for neuroregeneration in the adult mammalian central nervous system. However, many questions remain regarding how a terminally differentiated glial cell can transform into a delicate neuron that forms part of the intricate brain circuitry. In addition, concerns have recently been raised around the absence of astrocyte-to-neuron conversion in astrocytic lineage-tracing mice. In this study, we employed repetitive two-photon imaging to continuously capture the in situ astrocyte-to-neuron conversion process following ectopic expression of the neural transcription factor NeuroD1 in both proliferating reactive astrocytes and lineage-traced astrocytes in the mouse cortex. Time-lapse imaging over several weeks revealed the step-by-step transition from a typical astrocyte with numerous short, tapered branches to a typical neuron with a few long neurites and dynamic growth cones that actively explored the local environment. In addition, these lineage-converting cells were able to migrate radially or tangentially to relocate to suitable positions. Furthermore, two-photon Ca2+ imaging and patch-clamp recordings confirmed that the newly generated neurons exhibited synchronous calcium signals, repetitive action potentials, and spontaneous synaptic responses, suggesting that they had made functional synaptic connections within local neural circuits. In conclusion, we directly visualized the step-by-step lineage conversion process from astrocytes to functional neurons in vivo and unambiguously demonstrated that adult mammalian brains are highly plastic with respect to their potential for neuroregeneration and neural circuit reconstruction.

γδ T cells recruitment and local proliferation in brain parenchyma benefit anti-neuroinflammation after cerebral microbleeds.

Background: Cerebral microbleeds (CMBs) are an early sign of many neurological disorders and accompanied by local neuroinflammation and brain damage. As important regulators of immune response and neuroinflammation, the biological behavior and role of γδ T cells after CMBs remain largely unknown. Methods: We made a spot injury of microvessel in the somatosensory cortex to mimic the model of CMBs by two-photon laser and in vivo tracked dynamical behaviors of γδ T cells induced by CMBs using TCR-δGFP transgenic mice. Biological features of γδ T cells in the peri-CMBs parenchyma were decoded by flow cytometry and Raman spectra. In wildtype and γδ T cell-deficient mice, neuroinflammation and neurite degeneration in the peri-CMBs cortex were studied by RNAseq, immunostaining and in vivo imaging respectively. Results: After CMBs, γδ T cells in the dural vessels were tracked to cross the meningeal structure and invade the brain parenchyma in a few days, where the division process of γδ T cells were captured. Parenchymal γδ T cells were highly expressed by CXCR6 and CCR6, similar to meningeal γδ T cells, positive for IL-17A and Ki67 (more than 98%), and they contained abundant substances for energy metabolism and nucleic acid synthesis. In γδ T cell-deficient mice, cortical samples showed the upregulation of neuroinflammatory signaling pathways, enhanced glial response and M1 microglial polarization, and earlier neuronal degeneration in the peri-CMBs brain parenchyma compared with wildtype mice. Conclusion: CMBs induce the accumulation and local proliferation of γδ T cells in the brain parenchyma, and γδ T cells exert anti-neuroinflammatory and neuroprotective effects at the early stage of CMBs.

A Bright Monomeric Near-Infrared Fluorescent Protein with an Excitation Peak at 633 nm for Labeling Cellular Protein and Reporting Protein-Protein Interaction.

Bright monomeric near-infrared fluorescent proteins (NIR-FPs) are useful as markers for labeling proteins and cells and as sensors for reporting molecular activities in living cells and organisms. However, current monomeric NIR-FPs are dim under excitation with common 633/635/640 nm lasers, limiting their broad use in cellular/subcellular level imaging. Here, we report a bright monomeric NIR-FP with maximum excitation at 633 nm, named mIFP663, engineered from Xanthomonas campestris pv Campestris phytochrome (XccBphP). mIFP663 has high molecular brightness with a large extinction coefficient (86,600 M-1 cm-1) and a decent quantum yield (19.4%), and high cellular brightness that is 3-6 times greater than those of spectrally similar NIR-FPs in HEK293T cells in the presence of exogenous BV. Moreover, we demonstrate that mIFP663 is able to label critical cellular and viral proteins without perturbing subcellular localization and virus replication, respectively. Finally, with mIFP663, we engineer improved bimolecular fluorescence complementation (BiFC) and new bioluminescent resonance energy transfer (BRET) systems to detect protein-protein interactions in living cells.

Lineage tracing of direct astrocyte-to-neuron conversion in the mouse cortex.

Regenerating functional new neurons in the adult mammalian central nervous system has been proven to be very challenging due to the inability of neurons to divide and repopulate themselves after neuronal loss. Glial cells, on the other hand, can divide and repopulate themselves under injury or diseased conditions. We have previously reported that ectopic expression of NeuroD1 in dividing glial cells can directly convert them into neurons. Here, using astrocytic lineage-tracing reporter mice (Aldh1l1-CreERT2 mice crossing with Ai14 mice), we demonstrate that lineage-traced astrocytes can be successfully converted into NeuN-positive neurons after expressing NeuroD1 through adeno-associated viruses. Retroviral expression of NeuroD1 further confirms that dividing glial cells can be converted into neurons. Importantly, we demonstrate that for in vivo cell conversion study, using a safe level of adeno-associated virus dosage (1010-1012 gc/mL, 1 µL) in the rodent brain is critical to avoid artifacts caused by toxic dosage, such as that used in a recent bioRxiv study (2 × 1013 gc/mL, 1 µL, mouse cortex). For therapeutic purpose under injury or diseased conditions, or for non-human primate studies, adeno-associated virus dosage needs to be optimized through a series of dose-finding experiments. Moreover, for future in vivo glia-to-neuron conversion studies, we recommend that the adeno-associated virus results are further verified with retroviruses that mainly express transgenes in dividing glial cells in order to draw solid conclusions. The study was approved by the Laboratory Animal Ethics Committee of Jinan University, China (approval No. IACUC-20180330-06) on March 30, 2018.

Restoration of Visual Function and Cortical Connectivity After Ischemic Injury Through NeuroD1-Mediated Gene Therapy.

Neural circuits underlying brain functions are vulnerable to damage, including ischemic injury, leading to neuronal loss and gliosis. Recent technology of direct conversion of endogenous astrocytes into neurons in situ can simultaneously replenish the neuronal population and reverse the glial scar. However, whether these newly reprogrammed neurons undergo normal development, integrate into the existing neuronal circuit, and acquire functional properties specific for this circuit is not known. We investigated the effect of NeuroD1-mediated in vivo direct reprogramming on visual cortical circuit integration and functional recovery in a mouse model of ischemic injury. After performing electrophysiological extracellular recordings and two-photon calcium imaging of reprogrammed cells in vivo and mapping the synaptic connections formed onto these cells ex vivo, we discovered that NeuroD1 reprogrammed neurons were integrated into the cortical microcircuit and acquired direct visual responses. Furthermore, following visual experience, the reprogrammed neurons demonstrated maturation of orientation selectivity and functional connectivity. Our results show that NeuroD1-reprogrammed neurons can successfully develop and integrate into the visual cortical circuit leading to vision recovery after ischemic injury.

Lycium Barbarum Polysaccharides Protect Retina in rd1 Mice During Photoreceptor Degeneration.

Purpose: As an active component in wolfberry, lycium barbarum polysaccharides (LBP) are capable of protecting retinal neurons in several animal disease models. Here, we asked whether LBP rescues the retinal morphology and function in rd1 mouse, a photoreceptor fast-degenerating animal model of retinitis pigmentosa, and in particular focused on LBP's effects on the function of retinal ganglion cells (RGCs) during photoreceptor degeneration. Methods: An equal volume of LBP or control vehicle was daily intraperitoneal (i.p.) injected in rd1 mice from postnatal day 4 (P4) to P14, P20, or P24 when photoreceptors completely degenerate. Immunostaining, electroretinogram (ERG), visual behavior tests and multielectrode array (MEA) recordings were assessed to determine the structure and function of the treated retina. Results: LBP treatment greatly promoted photoreceptor survival, enhanced ERG responses, and improved visual behaviors in rd1 mice. MEA data showed that LBP treatment in general decreased the abnormally high spontaneous spiking that occurs in rd1 mice, and increased the percentage of light-responsive RGCs as well as their light-evoked response, light sensitivity, signal-to-noise ratio, and response speed. Interestingly, LBP treatment affected ON and OFF responses differently. Conclusions: LBP improves retinal morphology and function in rd1 mice, and delays the functional decay of RGCs during photoreceptor degeneration. This is the first study that has examined in detail the effects of LBP on RGC responses. Our data suggest that LBP may help extend the effective time window before more invasive RP therapeutic approaches such as retinoprosthesis are applied.

Inhibition of non-NMDA ionotropic glutamate receptors delays the retinal degeneration in rd10 mouse.

Retinitis pigmentosa (RP) is a hereditary blinding disease characterized by neurodegeneration of photoreceptors. Retinal ganglion cells (RGCs) in animal models of RP exhibit an abnormally high spontaneous activity that interferes with signal processing. Blocking AMPA/Kainate receptors by bath application of CNQX decreases the spontaneous firing, suggesting that inhibiting these receptors in vivo may help maintain the function of inner retinal neurons in rd10 mice experiencing photoreceptor degeneration. To test this, rd10 mice were i.p. injected with CNQX or GYKI 52466 (an AMPA receptor antagonist) for 1-2 weeks, and examined for their retinal morphology (by immunocytochemistry), function (by MEA recordings) and visual behaviors (using a black/white box). Our data show that iGluRs were up-regulated in the inner plexiform layer (IPL) of rd10 retinas. Application of CNQX at low doses both in vitro and in vivo, attenuated the abnormal spontaneous spiking in RGCs, and increased the light-evoked response of ON RGCs, whereas GYKI 52466 had little effect. CNQX application also improved the behavioral performance. Interestingly, in vivo administration of CNQX delayed photoreceptor degeneration, evidenced by the increased cell number and restored structure. CNQX also improved the structure of bipolar cells. Together, we demonstrated that during photoreceptor degeneration, blockade of the non-NMDA iGluRs decelerates the progression of RGCs dysfunction, possibly by dual mechanisms including slowing photoreceptor degeneration and modulating signal processing within the IPL. Accordingly, this strategy may effectively extend the time window for treating RP.