Parkinson's disease (PD) is the most common form of neurodegenerative movement disorder, associated with profound loss of dopaminergic neurons from the basal ganglia. Though loss of dopaminergic neuron cell bodies from the substantia nigra pars compacta is a well-studied feature, atrophy and loss of their axons within the nigrostriatal tract is also emerging as an early event in disease progression. Genes that drive the Wallerian degeneration, like Sterile alpha and toll/interleukin-1 receptor motif containing (Sarm1), are excellent candidates for driving this axon degeneration, given similarities in the morphology of axon degeneration after axotomy and in PD. In the present study we assessed whether Sarm1 contributes to loss of dopaminergic projections in mouse models of PD. In Sarm1 deficient mice, we observed a significant delay in the degeneration of severed dopaminergic axons distal to a 6-OHDA lesion of the medial forebrain bundle (MFB) in the nigrostriatal tract, and an accompanying rescue of morphological, biochemical and behavioural phenotypes. However, we observed no difference compared to controls when striatal terminals were lesioned with 6-OHDA to induce a dying back form of neurodegeneration. Likewise, when PD phenotypes were induced using AAV-induced alpha-synuclein overexpression, we observed similar modest loss of dopaminergic terminals in Sarm1 knockouts and controls. Our data argues that axon degeneration after MFB lesion is Sarm1-dependent, but that other models for PD do not require Sarm1, or that Sarm1 acts with other redundant genetic pathways. This work adds to a growing body of evidence indicating Sarm1 contributes to some, but not all types of neurodegeneration, and supports the notion that while axon degeneration in many context appears morphologically similar, a diversity of axon degeneration programs exist.

Although many features of embryonic development exhibit remarkable stability in the face of environmental perturbations, it is also clear that some aspects of early embryogenesis can be modulated by non-genetic influences during and after fertilization. Among potential perturbations experienced during reproduction, understanding the consequences of differing ex vivo fertilization methods at a molecular level is imperative for comprehending both the basic biology of early development and the potential consequences of assisted reproduction. Here, we set out to explore stable and flexible aspects of preimplantation gene expression using single-embryo RNA-sequencing of mouse embryos fertilized by natural mating, in vitro fertilization, or intracytoplasmic sperm injection, as well as oocytes parthenogenetically activated to develop (parthenotes). This dataset comprises a resource of over eight hundred individual embryos, which we use for three primary analyses. First, we characterize the effects of each fertilization method on early embryonic gene regulation, most notably finding decreased expression of trophectoderm markers at later stages of preimplantation development in ICSI embryos. Second, we find massive gene misregulation in parthenotes beyond the expected defects in imprinted gene expression, and show that many of these changes can be suppressed by sperm total RNA. Finally, we make use of the single-embryo resolution of our dataset to identify both stably-expressed genes and highly-variable genes in the early mouse embryo. Together, our data provide a detailed survey of the molecular consequences of different fertilization methods, establish parthenotes as a “tabula rasa” for understanding the role for sperm RNAs in preimplantation gene regulation, and identify subtypes of preimplantation embryos based on their expression of epivariable gene modules.

Zhou et al. (2019) report that embryos generated using sperm from the caput epididymis are fully capable of supporting full-term development, in contrast to our recent article (Conine et al., 2018). They also suggest that successful development of embryos generated using detergent-extracted sperm argues against the possibility that sperm delivers functional microRNAs (miRNAs) to the embryo. However, as several groups have demonstrated that sperm miRNAs are largely unaffected even by heavy detergent washes (Sharma et al., 2018,Yan et al., 2008), this experiment is not informative. We therefore focus on the primary finding of Zhou et al.: that embryos generated via intracytoplasmic sperm injection (ICSI) using caput epididymal sperm are viable and develop to term, which contrasts with Figures 3 and 7 of Conine et al., where we report that caput-derived embryos implant inefficiently and fail to develop further.

The small RNA payload of mammalian sperm undergoes dramatic remodeling during development, as several waves of microRNAs and tRNA fragments are shipped to sperm during post-testicular maturation in the epididymis. Here, we take advantage of this developmental process to probe the function of the sperm RNA payload in preimplantation development. We generated zygotes via intracytoplasmic sperm injection (ICSI) using sperm obtained from the proximal (caput) versus distal (cauda) epididymis and then characterized the development of the resulting embryos. Embryos generated using caput sperm significantly overexpress multiple regulatory factors throughout preimplantation development, subsequently implant inefficiently, and fail soon after implantation. Remarkably, microinjection of purified cauda-specific small RNAs into caput-derived embryos not only completely rescued preimplantation molecular defects but also suppressed the post-implantation embryonic lethality phenotype. These findings reveal an essential role for small RNA remodeling during post-testicular maturation of mammalian sperm and identify a specific preimplantation gene expression program responsive to sperm-delivered microRNAs.

Several recent studies link parental environments to phenotypes in subsequent generations. In this work, we investigate the mechanism by which paternal diet affects offspring metabolism. Protein restriction in mice affects small RNA (sRNA) levels in mature sperm, with decreased let-7 levels and increased amounts of 5' fragments of glycine transfer RNAs (tRNAs). In testicular sperm, tRNA fragments are scarce but increase in abundance as sperm mature in the epididymis. Epididymosomes (vesicles that fuse with sperm during epididymal transit) carry RNA payloads matching those of mature sperm and can deliver RNAs to immature sperm in vitro. Functionally, tRNA-glycine-GCC fragments repress genes associated with the endogenous retroelement MERVL, in both embryonic stem cells and embryos. Our results shed light on sRNA biogenesis and its dietary regulation during posttesticular sperm maturation, and they also link tRNA fragments to regulation of endogenous retroelements active in the preimplantation embryo.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease; survival in ALS is typically 3-5 years. No treatment extends patient survival by more than three months. Approximately 20% of familial ALS and 1-3% of sporadic ALS patients carry a mutation in the gene encoding superoxide dismutase 1 (SOD1). In a transgenic ALS mouse model expressing the mutant SOD1(G93A) protein, silencing the SOD1 gene prolongs survival. One study reports a therapeutic effect of silencing the SOD1 gene in systemically treated adult ALS mice; this was achieved with a short hairpin RNA, a silencing molecule that has raised multiple safety concerns, and recombinant adeno-associated virus (rAAV) 9. We report here a silencing method based on an artificial microRNA termed miR-SOD1 systemically delivered using adeno-associated virus rAAVrh10, a serotype with a demonstrated safety profile in CNS clinical trials. Silencing of SOD1 in adult SOD1(G93A) transgenic mice with this construct profoundly delayed both disease onset and death in the SOD1(G93A) mice, and significantly preserved muscle strength and motor and respiratory functions. We also document that intrathecal delivery of the same rAAVrh10-miR-SOD1 in nonhuman primates significantly and safely silences SOD1 in lower motor neurons. This study supports the view that rAAVrh10-miR-SOD1 merits further development for the treatment of SOD1-linked ALS in humans.

Alpha 1-Antitrypsin (AAT) deficiency is a human genetic disease resulting in the production of mutant AAT, a hepatocyte produced serine protease inhibitor that functions to prevent alveolar epithelial damage by inhibiting neutrophil elastase. Patients with AAT deficiency have increased lung disease, due to decreased proteolytic protection, as well as sporadic severe liver disease secondary to accumulation of mutant AAT, especially a common mutant form termed PiZ, within hepatocytes. We previously showed, in a PiZ mutant mouse model, simultaneous knock-down of mutant PiZ-AAT and augmentation of wild-type AAT production through intravenous delivery of a recombinant adeno-associated viral (rAAV) vector encoding both a miRNA targeting PiZ-AAT and a miRNA-resistant wild-type AAT gene. In this study we tested the hypothesis that rAAV2/9 vector administered intra-nasally or intra-tracheally can deliver a gene of interest to both the airways and liver. Initially C57Bl/6 mice were administered intra-nasally 1011 genome copies (GC) of rAAV2/9 vector expressing a firefly luciferase, which resulted in increased luminescence in the nasal passages, liver, and lung 21 days post delivery. Next, 1012 GC of rAAV2/9 vector expressing GFP and miRNAs targeting PiZ-AAT were delivered via oro-tracheal intubation to PiZ mice. This resulted in decreased serum AAT levels in the PiZ mice and GFP expression in both the liver and lungs. Finally, 1012 GC of rAAV2/9 vector encoding miRNA resistant wild-type AAT and miRNAs targeting PiZ-AAT were delivered via oro-tracheal intubation. This resulted in both systemic and local (liver and lung) elevations in wild-type AAT as well as decreased PiZ-AAT levels. In conclusion, tracheal delivery of rAAV2/9 resulted in expression of AAT in the liver and lung of treated animals, with sufficient targeting of the liver to mediate knock-down of mutant AAT to a similar degree as intravenous delivery, representing a potential non-invasive delivery route for gene therapy in AAT deficient patients.

Alpha 1-antitrypsin deficiency is a genetic disorder caused by defective production of alpha 1-antitrypsin (AAT). Gene therapy approaches have been conducted in patients with AAT deficiency with successful AAT expression, but not to the therapeutic levels required to reduce the risk of emphysema. Codon optimization, a somewhat new and evolving technique, is used by many scientists to maximize protein expression in living organisms by altering translational and transcriptional efficiency as well as protein refolding. The purpose of this study was to develop single stranded and double stranded AAT gene constructs, test their protein expression in vitro, and compare with those levels expressed by the AAT construct that is currently in clinical trials. Three constructs were to be developed, yet only one construct was successfully cloned. This clone, optimized ds-CB-AAT, illustrated increased AAT protein expression as the transfection time increased. However, protein levels were appreciably lower in the optimized construct compared to the single stranded (long intron) AAT construct that is currently being administered in clinical trials. The data did not suggest that the optimized AAT construct does in fact express more AAT protein in vitro as expected. In order to achieve data that can be reproduced, the 2 remaining constructs need to be cloned and all of the isolated plasmid DNA should be prepared on the same scale to minimize any additional confounding variables.

Alpha-1 antitrypsin (AAT) deficiency can exhibit two pathologic states: a lung disease that is primarily due to the loss of AAT's antiprotease function, and a liver disease resulting from a toxic gain-of-function of the PiZ-AAT (Z-AAT) mutant protein. We have developed several recombinant adeno-associated virus (rAAV) vectors that incorporate microRNA (miRNA) sequences targeting the AAT gene while also driving the expression of miRNA-resistant wild-type AAT-PiM (M-AAT) gene, thus achieving concomitant Z-AAT knockdown in the liver and increased expression of M-AAT. Transgenic mice expressing the human PiZ allele treated with dual-function rAAV9 vectors showed that serum PiZ was stably and persistently reduced by an average of 80%. Treated animals showed knockdown of Z-AAT in liver and serum with concomitant increased serum M-AAT as determined by allele-specific enzyme-linked immunosorbent assays (ELISAs). In addition, decreased globular accumulation of misfolded Z-AAT in hepatocytes and a reduction in inflammatory infiltrates in the liver was observed. Results from microarray studies demonstrate that endogenous miRNAs were minimally affected by this treatment. These data suggests that miRNA mediated knockdown does not saturate the miRNA pathway as has been seen with viral vector expression of short hairpin RNAs (shRNAs). This safe dual-therapy approach can be applied to other disorders such as amyotrophic lateral sclerosis, Huntington disease, cerebral ataxia, and optic atrophies.