Optineurin-facilitated axonal mitochondria delivery promotes neuroprotection and axon regeneration.

Optineurin (OPTN) mutations are linked to amyotrophic lateral sclerosis (ALS) and normal tension glaucoma (NTG), but a relevant animal model is lacking, and the molecular mechanisms underlying neurodegeneration are unknown. We found that OPTN C-terminus truncation (OPTN∆C) causes late-onset neurodegeneration of retinal ganglion cells (RGCs), optic nerve (ON), and spinal cord motor neurons, preceded by a striking decrease of axonal mitochondria. Surprisingly, we discover that OPTN directly interacts with both microtubules and the mitochondrial transport complex TRAK1/KIF5B, stabilizing them for proper anterograde axonal mitochondrial transport, in a C-terminus dependent manner. Encouragingly, overexpressing OPTN/TRAK1/KIF5B reverses not only OPTN truncation-induced, but also ocular hypertension-induced neurodegeneration, and promotes striking ON regeneration. Therefore, in addition to generating new animal models for NTG and ALS, our results establish OPTN as a novel facilitator of the microtubule-dependent mitochondrial transport necessary for adequate axonal mitochondria delivery, and its loss as the likely molecular mechanism of neurodegeneration.

Novel mutation in EFEMP1 identified from two Chinese POAG families differentially activated endoplasmic reticulum stress markers and induced glaucoma in mouse.

Primary open-angle glaucoma (POAG) is the most common type of glaucoma. Using whole-exome sequencing, we identified two independent families diagnosed as POAG from the China with a novel EFEMP1 variant (Exon3, c.175A>C p.Met59Leu); Three previously reported variants c.1160G>A p.R387Q, c.1189T>C p.Y397H, and c.1429C>T p.R477C in EFEPM1 from 55 sporadic POAG individuals were also identified. The variant c.175A>C p.Met59Leu co-segregated with the disease phenotype within the families. Immunoprecipitation and western blot assays showed that all three EFEMP1 mutants (p.Met59Leu, pArg140Trp, pArg345Trp) increased intracellular protein aggregations, and pMet59Leu and pArg140Arg also enhanced their extracellular proteins secretion, compared to WT in HEK293T. The differential regulations to endoplasmic reticulum (ER) stress markers ATF4, GPR78/94, and CHOP, and differential phosphorylation activations to CREB at Ser133, AKT at Ser473, p44/42 at Thr202/Tyr204, and STAT3 at Tyr705, were also detected among the mutants and WT. Finally, we revealed a significant increment of intraocular pressure and obvious reduction of RGC cells at the sixth week following intravitreal injection of adenovirus 5 (Ad5) expressing in pMet59Leu compared to WT and GFP controls. Together, variant c.175A>C p.Met59Leu in EFEMP1 is pathogenic and different mutants in EFEMP1 triggered distinct signaling pathways, explaining the reason of mutation-dependent disease phenotypes of EFEMP1.

Synthesis of Naphthalene- and Phenanthrene-Fused Smaragdyrins and Their BF2 Complexes.

Naphthalene- and phenanthrene-fused [22]smaragdyrin BF2 -complexes were synthesized by 1) Suzuki-Miyaura coupling of ß-brominated [22]smaragdyrin BF2 complexes with 2-formylarylboronates, 2) Witting-type methoxymethylenation of the formyl group, and 3) methanesulfonic acid-catalyzed cyclization reaction. Subsequently these BF2 complexes were deboronized and oxidized to the corresponding antiaromatic [20]smaragdyrin free bases. The installed fused structures led to decrease of the aromatic characters of the [22]smaragdyrin BF2 complexes and the antiaromatic characters of the [20]smaragdyrin free bases.

RGC-specific ATF4 and/or CHOP deletion rescues glaucomatous neurodegeneration and visual function.

Endoplasmic reticulum (ER) stress has been linked with various acute and chronic neurodegenerative diseases. We previously found that optic nerve (ON) injury and diseases induce neuronal ER stress in retinal ganglion cells (RGCs). We further demonstrated that germline deletion of CHOP preserves the structure and function of both RGC somata and axons in mouse glaucoma models. Here we report that RGC-specific deletion of CHOP and/or its upstream regulator ATF4 synergistically promotes RGC and ON survival and preserves visual function in mouse ON crush and silicone oil-induced ocular hypertension (SOHU) glaucoma models. Consistently, topical application of the ATF4/CHOP chemical inhibitor ISRIB or RGC-specific CRISPR-mediated knockdown of the ATF4 downstream effector Gadd45a also delivers significant neuroprotection in the SOHU glaucoma model. These studies suggest that blocking the neuronal intrinsic ATF4/CHOP axis of ER stress is a promising neuroprotection strategy for neurodegeneration.

The UPR Maintains Proteostasis and the Viability and Function of Hippocampal Neurons in Adult Mice.

The unfolded protein response (UPR), which comprises three branches: PERK, ATF6α, and IRE1, is a major mechanism for maintaining cellular proteostasis. Many studies show that the UPR is a major player in regulating neuron viability and function in various neurodegenerative diseases; however, its role in neurodegeneration is highly controversial. Moreover, while evidence suggests activation of the UPR in neurons under normal conditions, deficiency of individual branches of the UPR has no major effect on brain neurons in animals. It remains unclear whether or how the UPR participates in regulating neuronal proteostasis under normal and disease conditions. To determine the physiological role of the UPR in neurons, we generated mice with double deletion of PERK and ATF6α in neurons. We found that inactivation of PERK and ATF6α in neurons caused lysosomal dysfunction (as evidenced by decreased expression of the V0a1 subunit of v-ATPase and decreased activation of cathepsin D), impairment of autophagic flux (as evidenced by increased ratio of LC3-II/LC3-I and increased p62 level), and accumulation of p-tau and Aß42 in the hippocampus, and led to impairment of spatial memory, impairment of hippocampal LTP, and hippocampal degeneration in adult mice. These results suggest that the UPR is required for maintaining neuronal proteostasis (particularly tau and Aß homeostasis) and the viability and function of neurons in the hippocampus of adult mice.

Differential effects of SARM1 inhibition in traumatic glaucoma and EAE optic neuropathies.

Optic neuropathy is a group of optic nerve (ON) diseases with progressive degeneration of ON and retinal ganglion cells (RGCs). The lack of neuroprotective treatments is a central challenge for this leading cause of irreversible blindness. SARM1 (sterile α and TIR motif-containing protein 1) has intrinsic nicotinamide adenine dinucleotide (NAD+) hydrolase activity that causes axon degeneration by degrading axonal NAD+ significantly after activation by axon injury. SARM1 deletion is neuroprotective in many, but not all, neurodegenerative disease models. Here, we compare two therapy strategies for SARM1 inhibition, antisense oligonucleotide (ASO) and CRISPR, with germline SARM1 deletion in the neuroprotection of three optic neuropathy mouse models. This study reveals that, similar to germline SARM1 knockout in every cell, local retinal SARM1 ASO delivery and adeno-associated virus (AAV)-mediated RGC-specific CRISPR knockdown of SARM1 provide comparable neuroprotection to both RGC somata and axons in the silicone oil-induced ocular hypertension (SOHU) glaucoma model but only protect RGC axons, not somata, after traumatic ON injury. Surprisingly, neither of these two therapy strategies of SARM1 inhibition nor SARM1 germline knockout (KO) benefits RGC or ON survival in the experimental autoimmune encephalomyelitis (EAE)/optic neuritis model. Our studies therefore suggest that SARM1 inhibition by local ASO delivery or AAV-mediated CRISPR is a promising neuroprotective gene therapy strategy for traumatic and glaucomatous optic neuropathies but not for demyelinating optic neuritis.

Longitudinal in vivo Ca2+ imaging reveals dynamic activity changes of diseased retinal ganglion cells at the single-cell level.

Retinal ganglion cells (RGCs) are heterogeneous projection neurons that convey distinct visual features from the retina to brain. Here, we present a high-throughput in vivo RGC activity assay in response to light stimulation using noninvasive Ca2+ imaging of thousands of RGCs simultaneously in living mice. Population and single-cell analyses of longitudinal RGC Ca2+ imaging reveal distinct functional responses of RGCs and unprecedented individual RGC activity conversions during traumatic and glaucomatous degeneration. This study establishes a foundation for future in vivo RGC function classifications and longitudinal activity evaluations using more advanced imaging techniques and visual stimuli under normal, disease, and neural repair conditions. These analyses can be performed at both the population and single-cell levels using temporal and spatial information, which will be invaluable for understanding RGC pathophysiology and identifying functional biomarkers for diverse optic neuropathies.

Maprotiline restores ER homeostasis and rescues neurodegeneration via Histamine Receptor H1 inhibition in retinal ganglion cells.

When the protein or calcium homeostasis of the endoplasmic reticulum (ER) is adversely altered, cells experience ER stress that leads to various diseases including neurodegeneration. Genetic deletion of an ER stress downstream effector, CHOP, significantly protects neuron somata and axons. Here we report that three tricyclic compounds identified through a small-scale high throughput screening using a CHOP promoter-driven luciferase cell-based assay, effectively inhibit ER stress by antagonizing their common target, histamine receptor H1 (HRH1). We further demonstrated that systemic administration of one of these compounds, maprotiline, or CRISPR-mediated retinal ganglion cell (RGC)-specific HRH1 inhibition, delivers considerable neuroprotection of both RGC somata and axons and preservation of visual function in two mouse optic neuropathy models. Finally, we determine that maprotiline restores ER homeostasis by inhibiting HRH1-mediated Ca2+ release from ER. In this work we establish maprotiline as a candidate neuroprotectant and HRH1 as a potential therapeutic target for glaucoma.

Early Antimicrobial Evaluation of Nanostructured Surfaces Based on Bacterial Biological Properties.

Nanostructured physical antibacterial surfaces are of great interest due to the increasing antibiotic resistance. In this work, the titania nanotube (TNT) array, a potential physical antibacterial surface, was used for antimicrobial evaluation. The early antibacterial properties of TNTs were assessed based on three growth phases of Staphylococcus aureus (S. aureus), and the physical factors influencing the antibacterial properties were comprehensively discussed. The results show apparent early antibacterial effects of TNTs, including the anti-initial attachment during the lag phase, the inhibition of proliferation and bactericidal effect during the logarithmic phase, and the inhibition of biofilm formation during the stationary phase. These antimicrobial effects are closely related to the combined influence of various physical properties of TNTs, such as diameter, hydrophilicity, roughness, and charge. The present work suggests that the evaluation of the early antimicrobial behavior of biomaterials should pay more attention on the biological characteristics of bacteria.

Single-cell transcriptome analysis of regenerating RGCs reveals potent glaucoma neural repair genes.

Axon regeneration holds great promise for neural repair of CNS axonopathies, including glaucoma. Pten deletion in retinal ganglion cells (RGCs) promotes potent optic nerve regeneration, but only a small population of Pten-null RGCs are actually regenerating RGCs (regRGCs); most surviving RGCs (surRGCs) remain non-regenerative. Here, we developed a strategy to specifically label and purify regRGCs and surRGCs, respectively, from the same Pten-deletion mice after optic nerve crush, in which they differ only in their regeneration capability. Smart-Seq2 single-cell transcriptome analysis revealed novel regeneration-associated genes that significantly promote axon regeneration. The most potent of these, Anxa2, acts synergistically with its ligand tPA in Pten-deletion-induced axon regeneration. Anxa2, its downstream effector ILK, and Mpp1 dramatically protect RGC somata and axons and preserve visual function in a clinically relevant model of glaucoma, demonstrating the exciting potential of this innovative strategy to identify novel effective neural repair candidates.

Multiplexed genome regulation in vivo with hyper-efficient Cas12a.

Multiplexed modulation of endogenous genes is crucial for sophisticated gene therapy and cell engineering. CRISPR-Cas12a systems enable versatile multiple-genomic-loci targeting by processing numerous CRISPR RNAs (crRNAs) from a single transcript; however, their low efficiency has hindered in vivo applications. Through structure-guided protein engineering, we developed a hyper-efficient Lachnospiraceae bacterium Cas12a variant, termed hyperCas12a, with its catalytically dead version hyperdCas12a showing significantly enhanced efficacy for gene activation, particularly at low concentrations of crRNA. We demonstrate that hyperdCas12a has comparable off-target effects compared with the wild-type system and exhibits enhanced activity for gene editing and repression. Delivery of the hyperdCas12a activator and a single crRNA array simultaneously activating the endogenous Oct4, Sox2 and Klf4 genes in the retina of post-natal mice alters the differentiation of retinal progenitor cells. The hyperCas12a system offers a versatile in vivo tool for a broad range of gene-modulation and gene-therapy applications.

NMNAT2 is downregulated in glaucomatous RGCs, and RGC-specific gene therapy rescues neurodegeneration and visual function.

The lack of neuroprotective treatments for retinal ganglion cells (RGCs) and optic nerve (ON) is a central challenge for glaucoma management. Emerging evidence suggests that redox factor NAD+ decline is a hallmark of aging and neurodegenerative diseases. Supplementation with NAD+ precursors and overexpression of NMNAT1, the key enzyme in the NAD+ biosynthetic process, have significant neuroprotective effects. We first profile the translatomes of RGCs in naive mice and mice with silicone oil-induced ocular hypertension (SOHU)/glaucoma by RiboTag mRNA sequencing. Intriguingly, only NMNAT2, but not NMNAT1 or NMNAT3, is significantly decreased in SOHU glaucomatous RGCs, which we confirm by in situ hybridization. We next demonstrate that AAV2 intravitreal injection-mediated overexpression of long half-life NMNAT2 mutant driven by RGC-specific mouse γ-synuclein (mSncg) promoter restores decreased NAD+ levels in glaucomatous RGCs and ONs. Moreover, this RGC-specific gene therapy strategy delivers significant neuroprotection of both RGC soma and axon and preservation of visual function in the traumatic ON crush model and the SOHU glaucoma model. Collectively, our studies suggest that the weakening of NMNAT2 expression in glaucomatous RGCs contributes to a deleterious NAD+ decline, and that modulating RGC-intrinsic NMNAT2 levels by AAV2-mSncg vector is a promising gene therapy for glaucomatous neurodegeneration.

Neuronal NMNAT2 Overexpression Does Not Achieve Significant Neuroprotection in Experimental Autoimmune Encephalomyelitis/Optic Neuritis.

Optic neuritis, inflammation, and demyelination of the optic nerve (ON), is one of the most common clinical manifestations of multiple sclerosis; affected patients suffer persistent visual symptoms due to ON degeneration and secondary retinal ganglion cell (RGC) death. The mouse experimental autoimmune encephalomyelitis (EAE) model replicates optic neuritis and significant RGC soma and axon loss. Nicotinamide mononucleotide adenylyltransferases (NMNATs) are NAD+-synthetic enzymes that have been shown to be essential for axon integrity, activation of which significantly delays axonal Wallerian degeneration. NMNAT2, which is enriched in axons, has been proposed as a promising therapeutic target for axon injury-induced neurodegeneration. We therefore investigated whether activation of NMNAT2 can be used as a gene therapy strategy for neuroprotection in EAE/optic neuritis. To avoid the confounding effects in inflammatory cells, which play important roles in EAE initiation and progression, we used an RGC-specific promoter to drive the expression of the long half-life NMNAT2 mutant in mouse RGCs in vivo. However, optical coherence tomography in vivo retina imaging did not reveal significant protection of the ganglion cell complex, and visual function assays, pattern electroretinography, and optokinetic response also showed no improvement in mice with NMNAT2 overexpression. Postmortem histological analysis of retina wholemounts and semithin sections of ON confirmed the in vivo results: NMNAT2 activation in RGCs does not provide significant neuroprotection of RGCs in EAE/optic neuritis. Our studies suggest that a different degenerative mechanism than Wallerian degeneration is involved in autoimmune inflammatory axonopathy and that NMNAT2 may not be a major contributor to this mechanism.

Chronic mild and acute severe glaucomatous neurodegeneration derived from silicone oil-induced ocular hypertension.

Recently, we established silicone oil-induced ocular hypertension (SOHU) mouse model with significant glaucomatous neurodegeneration. Here we characterize two additional variations of this model that simulate two distinct glaucoma types. The first is a chronic model produced by high frequency (HF) pupillary dilation after SO-induced pupillary block, which shows sustained moderate IOP elevation and corresponding slow, mild glaucomatous neurodegeneration. We also demonstrate that although SO removal quickly returns IOP to normal, the glaucomatous neurodegeneration continues to advance to a similar degree as in the HF group without SO removal. The second, an acute model created by no pupillary dilation (ND), shows a greatly elevated IOP and severe inner retina degeneration at an early time point. Therefore, by a straightforward dilation scheme, we extend our original SOHU model to recapitulate phenotypes of two major glaucoma forms, which will be invaluable for selecting neuroprotectants and elucidating their molecular mechanisms.

A Robust Porphyrin-Stabilized Triplet Carbon Diradical.

The synthesis of robust high-spin carbon radicals is an important topic in organic chemistry. Toward this end, several porphyrin-stabilized radicals have been systematically explored. A singly naphthalene-fused porphyrin radical was synthesized by a reaction sequence consisting of a Suzuki-Miyaura coupling of ß-borylated porphyrin with 2-bromobenzaldehyde, addition of mesityl Grignard reagent, intramolecular Friedel-Crafts alkylation, and final oxidation with DDQ or tBuOK/O2 . This strategy was also used to synthesize doubly naphthalene-fused porphyrins and syn- and anti-fused-anthracene-bridged porphyrin dimers. While singly naphthalene-fused porphyrin radical has been shown to be a stable monoradical, doubly naphthalene-fused porphyrins and anti-fused-anthracene-bridged porphyrin dimers have been shown to be closed-shell molecules. Finally, the syn-dimer was characterized as a surprisingly stable radical (t1/2 =28 days under ambient air and at 80 °C) that is storable for more than several months, despite its high-spin triplet ground-state carbon diradical.

Alpha 1-antitrypsin ameliorates ventilator-induced lung injury in rats by inhibiting inflammatory responses and apoptosis.

Mechanical ventilation is extensively used to treat patients with lung injury but may result in ventilator-induced lung injury (VILI). The present study investigated the protective effect of alpha 1-antitrypsin (AAT) on VILI. Adult male rats were subjected to sham, ventilation + saline, or ventilation + AAT treatment and lung injuries were evaluated. Peripheral blood and bronchoalveolar lavage fluid (BALF) were obtained to assess systemic and local inflammatory responses, respectively. Mechanical ventilation resulted in lung injury, as evidenced by histological abnormalities as well as elevations in PaO2/FiO2 ratio, the wet-to-dry weight ratio, and the BALF level of proteins. The intravenous administration of AAT significantly improved these parameters of lung function, suggesting a protective role of AAT in VILI. Mechanistically, ventilator-induced inflammation was effectively reduced by AAT, as evidenced by decreases in BALF neutrophil counts, BALF cytokines, and serum adhesion factors. In contrast, anti-inflammatory interleukin-10 in BALF was increased in response to AAT. AAT treatment also inhibited the expression of nuclear factor-κB, Bax, and cleaved caspase-3 while promoting Bcl-2 expression in ventilator-injured lung tissues. AAT treatment can ameliorate VILI by inhibiting inflammatory mediator production and apoptosis. Impact statement Mechanical ventilation has been commonly used to treat patients with lung injury but may result in ventilator-induced lung injury (VILI). Few effective treatment options are currently available to reduce VILI. Alpha 1-antitrypsin (AAT) is an inhibitor of serine protease with anti-inflammatory and antiapoptotic properties, suggesting a possible role in attenuating lung injury. The present study demonstrates that AAT inhibits the development of VILI by modulating inflammation- and apoptosis-related protein expression. Therefore, AAT may be a novel therapeutic agent for acute respiratory distress syndrome patients undergoing mechanical ventilation.

Proteomic analysis of the cullin 4B interactome using proximity-dependent biotinylation in living cells.

Cullin 4B (CUL4B) mutations have been implicated in mental retardation and dopamine-related behaviors due to disruptions in their interaction with cullin-RING E3 ligases (CRLs). Thus, further identification of CUL4B substrates can increase the knowledge of protein homeostasis and illuminate the role of CUL4B in neuropsychiatric disease. However, the transient nature of the coupling between CUL4B and its substrates is difficult to detect in vivo using current approaches, thus hampers efforts to investigate functions of CRLs within unperturbed living systems. In this study, we sought to discover CUL4B interactants with or without dopamine stimulation. BirA (118G) proximity-dependent biotin labeling combined with LC-MS was employed to biotinylate and identify transient and weak interactants of CUL4B. After purification with streptavidin beads and identified by LC-MS, a total of 150 biotinylated proteins were identified at baseline condition, 53 of which are well-known CUL4B interactants. After dopamine stimulation, 29 proteins disappeared and were replaced by 21 different protein interactants. The altered CUL4B interactants suggest that CUL4B regulates protein turnover and homeostasis in response to dopamine stimulation. Our results demonstrate the potential of this approach to identify novel CUL4B-related molecules in respond to cellular stimuli, which may be applied to other types of signaling pathways.

S113R mutation in SLC33A1 leads to neurodegeneration and augmented BMP signaling in a mouse model.

The S113R mutation (c.339T>G) (MIM #603690.0001) in SLC33A1 (MIM #603690), an ER membrane acetyl-CoA transporter, has been previously identified in individuals with hereditary spastic paraplegia type 42 (SPG42; MIM #612539). SLC33A1 has also been shown to inhibit the bone morphogenetic protein (BMP) signaling pathway in zebrafish. To better understand the function of SLC33A1, we generated and characterized Slc33a1S113R knock-in mice. Homozygous Slc33a1S113R mutant mice were embryonic lethal, whereas heterozygous Slc33a1 mutant mice (Slc33a1wt/mut) exhibited behavioral abnormalities and central neurodegeneration, which is consistent with hereditary spastic paraplegia (HSP) phenotypes. Importantly, we found an upregulation of BMP signaling in the nervous system and mouse embryonic fibroblasts of Slc33a1wt/mut mice. Using a sciatic nerve crush injury model in vivo and dorsal root ganglion (DRG) culture in vitro we showed that injury-induced axonal regeneration in Slc33a1wt/mut mice was accelerated and mediated by upregulated BMP signaling. Exogenous addition of BMP signaling antagonist, noggin, could efficiently alleviate the accelerated injury-induced axonal regrowth. These results indicate that SLC33A1 can negatively regulate BMP signaling in mice, further supporting the notion that upregulation of BMP signaling is a common mechanism of a subset of hereditary spastic paraplegias.

Identification and functional analysis of a SLC33A1: c.339T>G (p.Ser113Arg) variant in the original SPG42 family.

Using whole-exome sequencing, we surveyed all the potential pathogenic variants in an SPG42 family and found five SNPs and four indels that are shared by two patients and lie in the mapped region. Two variants, SLC33A1 p.Ser113Arg and VEPH1 p.Gln433His, cosegregated with the disease. However, VEPH1 p.Gln433His was predicted to be tolerated, thus leaving SLC33A1 p.Ser113Arg as the most plausible causal variant in this family. We found that the phosphorylated SMAD1/5/8 (P-SMAD1/5/8) and BMP receptor type 1A (BMPR1A) were substantially upregulated in fibroblasts derived from an SPG42 individual. Slc33a1 knockdown zebrafish, which exhibited defects in morphology and axon outgrowth, also showed a significant elevation in the level of P-smad1/5/8. While the phenotypes in slc33a1 knockdown zebrafish could be rescued by human wild-type SLC33A1 mRNA, this rescuing effect was diminished by coinjected mutant mRNA encoding p.Ser113Arg, indicating that p.Ser113Arg variant acts in a dominant-negative manner. Importantly, pharmacological blockade of BMPR1 activity by dorsomorphin could efficiently rescue the phenotypic defects in slc33a1 knockdown zebrafish. These results indicate that SLC33A1 can negatively regulate BMP signaling.

Synthesis and performance evaluation of a new deoiling agent for treatment of waste oil-based drilling fluids.

Oil-based drilling fluid is used more and more in the field of oil and gas exploration. However, because of unrecyclable treating agent and hard treatment conditions, the traditional treating technologies of waste oil-based drilling fluid have some defects, such as waste of resource, bulky equipment, complex treatment processes, and low oil recovery rate. In this work, switchable deoiling agent (SDA), as a novel surfactant for treatment of waste oil-based drilling fluid, was synthesized by amine, formic acid, and formaldehyde solution. With this agent, the waste oil-based drilling fluid can be treated without complex process and expensive equipment. Furthermore, the agent used in the treatment can be recycled, which reduces waste of resource and energy. The switch performance, deoiling performance, structural characterization, and mechanisms of action are studied. The experimental results show that the oil content of the recycled oil is higher than 96% and more than 93% oil in waste oil-based drilling fluid can be recycled. The oil content of the solid residues of deoiling is less than 3%.