Controlling donor and newborn neuron migration and maturation in the eye through microenvironment engineering.

Ongoing cell therapy trials have demonstrated the need for precision control of donor cell behavior within the recipient tissue. We present a methodology to guide stem cell-derived and endogenously regenerated neurons by engineering the microenvironment. Being an "approachable part of the brain," the eye provides a unique opportunity to study neuron fate and function within the central nervous system. Here, we focused on retinal ganglion cells (RGCs)-the neurons in the retina are irreversibly lost in glaucoma and other optic neuropathies but can potentially be replaced through transplantation or reprogramming. One of the significant barriers to successful RGC integration into the existing mature retinal circuitry is cell migration toward their natural position in the retina. Our in silico analysis of the single-cell transcriptome of the developing human retina identified six receptor-ligand candidates, which were tested in functional in vitro assays for their ability to guide human stem cell-derived RGCs. We used our lead molecule, SDF1, to engineer an artificial gradient in the retina, which led to a 2.7-fold increase in donor RGC migration into the ganglion cell layer (GCL) and a 3.3-fold increase in the displacement of newborn RGCs out of the inner nuclear layer. Only donor RGCs that migrated into the GCL were found to express mature RGC markers, indicating the importance of proper structure integration. Together, these results describe an "in silico-in vitro-in vivo" framework for identifying, selecting, and applying soluble ligands to control donor cell function after transplantation.

Identification of precursor microRNAs within distal axons of sensory neuron.

A set of specific precursor microRNAs (pre-miRNAs) are reported to localize into neuronal dendrites, where they could be processed locally to control synaptic protein synthesis and plasticity. However, it is not clear whether specific pre-miRNAs are also transported into distal axons to autonomously regulate intra-axonal protein synthesis. Here, we show that a subset of pre-miRNAs, whose mature miRNAs are enriched in axonal compartment of sympathetic neurons, are present in axons of neurons both in vivo and in vitro by quantitative PCR and by in situ hybridization. Some pre-miRNAs (let 7c-a and pre-miRs-16, 23a, 25, 125b-1, 433, and 541) showed elevated axonal levels, while others (pre-miRs-138-2, 185, and 221) were decreased in axonal levels following injury. Dicer and KSRP proteins are also present in distal axons, but Drosha is found restricted to the cell body. These findings suggest that specific pre-miRNAs are selected for localization into distal axons of sensory neurons and are presumably processed to mature miRNAs in response to extracellular stimuli. This study supports the notion that local miRNA biogenesis effectively provides another level of temporal control for local protein synthesis in axons.

Sustained neurotrophic factor cotreatment enhances donor and host retinal ganglion cell survival in mice.

Retinal ganglion cells (RGCs) lack regenerative capacity in mammals, and their degeneration in glaucoma leads to irreversible blindness. The transplantation of stem cell-derived RGCs lacks clinically relevant effect due to insufficient survival and integration of donor cells. We hypothesize that the retinal microenvironment plays a critical role in this process, and we can engineer a more acceptable setting for transplantation. Since the adult mammalian retina does not have regenerative capacity, we turned to the developing human retina to reconstruct cell-cell interactions at a single-cell level. We established a human fetal retina atlas by integrating currently available single-cell RNA sequencing datasets of human fetal retinas into a unified resource. We align RGC transcriptomes in pseudotime to map RGC developmental fate trajectories against the broader timeline of retinal development. Through this analysis, we identified brain-derived neurotrophic factor (BDNF) and glial-derived neurotrophic factor (GDNF) as key factors in RGC survival, highly expressed during fetal development but significantly reduced in adulthood despite the persistence of their receptors. To demonstrate the practical application of these findings, we show that using a slow-release formulation of BDNF and GDNF enhances RGC differentiation, survival, and function in vitro and improves RGC transplantation outcomes in a mouse model. BNDF/GDNF co-treatment not only increased survival and coverage of donor RGCs within the retina but also showed neuroprotective effects on host RGCs, preserving retinal function in a model of optic neuropathy. Altogether, our findings suggest that manipulating the retinal microenvironment with slow-release neurotrophic factors holds promise in regenerative medicine for treating glaucoma and other optic neuropathies. This approach not only improves donor cell survival and integration but also provides a neuroprotective benefit to host cells, indicating a significant advancement for glaucoma therapies.

Effects of iron depletion on Entamoeba histolytica alcohol dehydrogenase 2 (EhADH2) and trophozoite growth: implications for antiamoebic therapy.

OBJECTIVES: The purpose of this study was to determine the mechanism by which iron chelation affects the trophozoite survival of Entamoeba histolytica. Fe2+ is a cofactor for E. histolytica alcohol dehydrogenase 2 (EhADH2), an essential bifunctional enzyme [alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH)] in the glycolytic pathway of E. histolytica. METHODS: We tested the effects of iron depletion on trophozoite growth, the kinetics of iron binding to EhADH2, and the activities of ADH and ALDH. RESULTS: Growth of E. histolytica trophozoites, and ADH and ALDH enzymatic activities were directly inhibited by iron chelation. Kinetics of iron binding to EhADH2 reveals the differential iron affinity of ADH (higher) and ALDH (lower). CONCLUSIONS: This study demonstrates that iron chelation interrupts the completion of the fermentative pathway of E. histolytica by removing the metal cofactor indispensable for the structural and functional stability of EhADH2, thus affecting trophozoite survival. We propose that iron-starvation-based strategies could be used to treat amoebiasis.

Differential regulation of brain-derived neurotrophic factor (BDNF) expression in sensory neuron axons by miRNA-206.

Distinct subcellular localization and subsequent translational control of 3' UTR variants of mRNA encoding brain-derived neurotrophic factor (BDNF) are critical for the development and plasticity of neurons. Although the processes that lead to preferential localization of BDNF have been well studied, it is still not clear how neurons ensure differential BDNF production in a spatial-specific manner. Here, we identified that microRNA (miRNA)-206 has the potential to specifically regulate BDNF with a long 3' UTR without affecting its short 3' UTR counterpart. Overexpression of miRNA-206 in sensory neurons resulted in a 30% and 45% reduction of BDNF protein expression in the cell bodies and axons, respectively. The work described in the present study indicates that miRNAs can differentially and specifically regulate the expression of transcript variants with different localization patterns.

Analysis of piRNA-Like Small Non-coding RNAs Present in Axons of Adult Sensory Neurons.

Small non-coding RNAs (sncRNAs) have been shown to play pivotal roles in spatiotemporal-specific gene regulation that is linked to many different biological functions. PIWI-interacting RNAs (piRNAs), typically 25-34-nucleotide long, are originally identified and thought to be restricted in germline cells. However, recent studies suggest that piRNAs associate with neuronal PIWI proteins, contributing to neuronal development and function. Here, we identify a cohort of piRNA-like sncRNAs (piLRNAs) in rat sciatic nerve axoplasm and directly contrast temporal changes of piLRNA levels in the nerve following injury, as compared with those in an uninjured nerve using deep sequencing. We find that 32 of a total of 53 annotated piLRNAs show significant changes in their levels in the regenerating nerve, suggesting that individual axonal piLRNAs may play important regulatory roles in local messenger RNA (mRNA) translation during regeneration. Bioinformatics and biochemical analyses show that these piLRNAs carry characteristic features of mammalian piRNAs, including sizes, a sequence bias for uracil at the 5'-end and a 2'-O-methylation at the 3'-end. Their axonal expression is directly visualized by fluorescence in situ hybridization in cultured dorsal root ganglion neurons as well as immunoprecipitation with MIWI. Further, depletion of MIWI protein using RNAi from cultured sensory neurons increases axon growth rates, decreases axon retraction after injury, and increases axon regrowth after injury. All these data suggest more general roles for MIWI/piLRNA pathway that could confer a unique advantage for coordinately altering the population of proteins generated in growth cones and axons of neurons by targeting mRNA cohorts.

Dynamic Change and Target Prediction of Axon-Specific MicroRNAs in Regenerating Sciatic Nerve.

Injury to axons in the peripheral nervous system induces rapid and local regenerative responses to form a new growth cone, and to generate a retrogradely transporting injury signal. The evidence for essential roles of intra-axonal protein synthesis during regeneration is now compelling. MicroRNA (miRNA) has recently been recognized as a prominent player in post-transcriptional regulation of axonal protein synthesis. Here, we directly contrast temporal changes of miRNA levels in the sciatic nerve following injury, as compared to those in an uninjured nerve using deep sequencing. Small RNAs (<200 nucleotides in length) were fractionated from the proximal nerve stumps to improve the representation of differential miRNA levels. Of 141 axoplasmic miRNAs annotated, 63 rat miRNAs showed significantly differential levels at five time points following injury, compared to an uninjured nerve. The differential changes in miRNA levels responding to injury were processed for hierarchical clustering analyses, and used to predict target mRNAs by Targetscan and miRanda. By overlapping these predicted targets with 2,924 axonally localizing transcripts previously reported, the overlapping set of 214 transcripts was further analyzed by the Gene Ontology enrichment and Ingenuity Pathway Analyses. These results suggest the possibility that the potential targets for these miRNAs play key roles in numerous neurological functions involved in ER stress response, cytoskeleton dynamics, vesicle formation, and neuro-degeneration and-regeneration. Finally, our results suggest that miRNAs could play a direct role in regenerative response and may be manipulated to promote regenerative ability of injured nerves.

IgG Conformer's Binding to Amyloidogenic Aggregates.

Amyloid-reactive IgGs isolated from pooled blood of normal individuals (pAbs) have demonstrated clinical utility for amyloid diseases by in vivo targeting and clearing amyloidogenic proteins and peptides. We now report the following three novel findings on pAb conformer's binding to amyloidogenic aggregates: 1) pAb aggregates have greater activity than monomers (HMW species > dimers > monomers), 2) pAbs interactions with amyloidogenic aggregates at least partially involves unconventional (non-CDR) interactions of F(ab) regions, and 3) pAb's activity can be easily modulated by trace aggregates generated during sample processing. Specifically, we show that HMW aggregates and dimeric pAbs present in commercial preparations of pAbs, intravenous immunoglobulin (IVIg), had up to ~200- and ~7-fold stronger binding to aggregates of Aß and transthyretin (TTR) than the monomeric antibody. Notably, HMW aggregates were primarily responsible for the enhanced anti-amyloid activities of Aß- and Cibacron blue-isolated IVIg IgGs. Human pAb conformer's binding to amyloidogenic aggregates was retained in normal human sera, and mimicked by murine pAbs isolated from normal pooled plasmas. An unconventional (non-CDR) component to pAb's activity was indicated from control human mAbs, generated against non-amyloid targets, binding to aggregated Aß and TTR. Similar to pAbs, HMW and dimeric mAb conformers bound stronger than their monomeric forms to amyloidogenic aggregates. However, mAbs had lower maximum binding signals, indicating that pAbs were required to saturate a diverse collection of binding sites. Taken together, our findings strongly support further investigations on the physiological function and clinical utility of the inherent anti-amyloid activities of monomeric but not aggregated IgGs.

Transthyretin aggregate-specific antibodies recognize cryptic epitopes on patient-derived amyloid fibrils.

Amyloidosis involves the extracellular deposition of proteinaceous amyloid fibrils and accessory molecules in organ(s) and/or tissue(s), and is associated with a host of human diseases, including Alzheimer disease, diabetes, and heart disease. Unfortunately, the amyloidoses are currently incurable, and there is an urgent need for less invasive diagnostics. To address this, we have generated 22 monoclonal antibodies (mAbs) against aggregates formed by a blood transport protein, transthyretin (TTR), which primarily forms amyloid fibrils in a patient's heart and/or peripheral nerves. Four of the mAbs, 2T5C9, 2G9C, T1F11, and TB2H7, demonstrated diagnostic potential in enzyme-linked immunosorbent assays (ELISA) by their low to sub-nanomolar cross-reactivity with recombinant wild-type (WT) and mutant TTR aggregates and lack of binding to native TTR or amyloid fibrils formed by other peptides or proteins. Notably, in the presence of normal human sera, three of the four mAbs, 2T5C9, 2G9C, and T1F11, retained low nM binding to TTR amyloid fibrils derived from two patients with familial amyloidotic polyneuropathy (FAP). The two most promising mAbs, 2T5C9 and 2G9C, were also shown by immunohistochemistry to have low nM binding to TTR amyloid deposits in cardiac tissue sections from two FAP patients. Taken together, these findings strongly support further investigations on the diagnostic utility of TTR aggregate specific mAbs for patients with TTR amyloidoses.