POSTINJURY FECAL MICROBIOME TRANSPLANT DECREASES LESION SIZE AND NEUROINFLAMMATION IN TRAUMATIC BRAIN INJURY.

ABSTRACT: Background: Traumatic brain injury (TBI) is an underrecognized public health threat. The constitutive activation of microglia after TBI has been linked to long-term neurocognitive deficits and the progression of neurodegenerative disease. Evolving evidence indicates a critical role for the gut-brain axis in this process. Specifically, TBI has been shown to induce the depletion of commensal gut bacteria. The resulting gut dysbiosis is associated with neuroinflammation and disease. Hypothesis: We hypothesized that fecal microbiota transplantation would attenuate microglial activation and improve neuropathology after TBI. Methods: C57Bl/6 mice were subjected to severe TBI (n = 10) or sham injury (n = 10) via an open-head controlled cortical impact. The mice underwent fecal microbiota transplantation (FMT) or vehicle alone via oral gavage once weekly for 4 weeks after injury. At 59 days after TBI, mice underwent three-dimensional, contrast-enhanced magnetic resonance imaging. Following imaging, mice were killed, brains harvested at 60 DPI, and CD45+ cells isolated via florescence-activated cell sorting. cDNA libraries were prepared using the 10x Genomics Chromium Single Cell 3' Reagent kit followed by sequencing on a HiSeq4000 instrument, and computational analysis was performed. Results: Fecal microbiota transplantation resulted in a >marked reduction of ventriculomegaly (P < 0.002) and preservation of white matter connectivity at 59 days after TBI (P < 0.0001). In addition, microglia from FMT-treated mice significantly reduced inflammatory gene expression and enriched pathways involving the heat-shock response compared with mice treated with vehicle alone. Conclusions: We hypothesized that restoring gut microbial community structure via FMT would attenuate microglial activation and reduce neuropathology after TBI. Our data demonstrated significant preservation of cortical volume and white matter connectivity after an injury compared with mice treated with vehicle alone. This preservation of neuroanatomy after TBI was associated with a marked reduction in inflammatory gene expression within the microglia of FMT-treated mice. Microglia from FMT-treated mice enriched pathways in the heat-shock response, which is known to play a neuroprotective role in TBI and other neurodegenerative disease processes.

Sertoli cells require TDP-43 to support spermatogenesis†.

TAR DNA binding protein of 43 kD (TDP-43) is an evolutionarily conserved, ubiquitously expressed transcription factor and RNA-binding protein with major human health relevance. TDP-43 is present in Sertoli and germ cells of the testis and is aberrantly expressed in the sperm of infertile men. Sertoli cells play a key role in spermatogenesis by offering physical and nutritional support to male germ cells. The current study investigated the requirement of TDP-43 in Sertoli cells. Conditional knockout (cKO) of TDP-43 in mouse Sertoli cells caused failure of spermatogenesis and male subfertility. The cKO mice showed decreased testis weight, and low sperm count. Testis showed loss of germ cell layers, presence of vacuoles, and sloughing of round spermatids, suggesting loss of contact with Sertoli cells. Using a biotin tracer, we found that the blood-testis barrier (BTB) was disrupted as early as postnatal day 24 and worsened in adult cKO mice. We noted aberrant expression of the junction proteins connexin-43 (gap junction) and N-cadherin (ectoplasmic specialization). Oil Red O staining showed a decrease in lipid droplets (phagocytic function) in tubule cross-sections, Sertoli cells cytoplasm, and in the lumen of seminiferous tubules of cKO mice. Finally, qRT-PCR showed upregulation of genes involved in the formation and/or maintenance of Sertoli cell junctions as well as in the phagocytic pathway. Sertoli cells require TDP-43 for germ cell attachment, formation and maintenance of BTB, and phagocytic function, thus indicating an essential role for TDP-43 in the maintenance of spermatogenesis.

Fecal Microbiota Transfer Attenuates Gut Dysbiosis and Functional Deficits After Traumatic Brain Injury.

BACKGROUND: Traumatic brain injury (TBI) is an underrecognized public health threat. Survivors of TBI often suffer long-term neurocognitive deficits leading to the progressive onset of neurodegenerative disease. Recent data suggests that the gut-brain axis is complicit in this process. However, no study has specifically addressed whether fecal microbiota transfer (FMT) attenuates neurologic deficits after TBI. HYPOTHESIS: We hypothesized that fecal microbiota transfer would attenuate neurocognitive, anatomic, and pathologic deficits after TBI. METHODS: C57Bl/6 mice were subjected to severe TBI (n = 20) or sham-injury (n = 20) via an open-head controlled cortical impact. Post-injury, this cohort of mice underwent weekly oral gavage with a slurry of healthy mouse stool or vehicle alone beginning 1 h post-TBI followed by behavioral testing and neuropathologic analysis. 16S ribosomal RNA sequencing of fecal samples was performed to characterize gut microbial community structure pre- and post-injury. Zero maze and open field testing were used to evaluate post-traumatic anxiety, exploratory behavior, and generalized activity. 3D, contrast enhanced, magnetic resonance imaging was used to determine differences in cortical volume loss and white matter connectivity. Prior to euthanasia, brains were harvested for neuropathologic analysis. RESULTS: Fecal microbiome analysis revealed a large variance between TBI, and sham animals treated with vehicle, while FMT treated TBI mice had restoration of gut dysbiosis back to levels of control mice. Neurocognitive testing demonstrated a rescue of normal anxiety-like and exploratory behavior in TBI mice treated with FMT. FMT treated TBI mice spent a greater percentage of time (22%, P = 0.0001) in the center regions of the Open Field as compared to vehicle treated TBI mice (13%). Vehicle-treated TBI animals also spent less time (19%) in the open areas of zero maze than FMT treated TBI mice (30%, P = 0.0001). Comparing in TBI mice treated with FMT, MRI demonstrated a marked attenuation in ventriculomegaly (P < 0.002) and a significant change in fractional anisotropy (i.e., loss of white matter connectivity) (P < 0.0001). Histologic analysis of brain sections revealed a FMT- injury dependent interaction in the microglia/macrophage-specific ionized calcium-binding protein, Iba1 (P = 0.002). CONCLUSION: These data suggest that restoring a pre-injury gut microbial community structure may be a promising therapeutic intervention after TBI.

Loss of TDP-43 in male germ cells causes meiotic failure and impairs fertility in mice.

Meiotic arrest is a common cause of human male infertility, but the causes of this arrest are poorly understood. Transactive response DNA-binding protein of 43 kDa (TDP-43) is highly expressed in spermatocytes in the preleptotene and pachytene stages of meiosis. TDP-43 is linked to several human neurodegenerative disorders wherein its nuclear clearance accompanied by cytoplasmic aggregates underlies neurodegeneration. Exploring the functional requirement for TDP-43 for spermatogenesis for the first time, we show here that conditional KO (cKO) of the Tardbp gene (encoding TDP-43) in male germ cells of mice leads to reduced testis size, depletion of germ cells, vacuole formation within the seminiferous epithelium, and reduced sperm production. Fertility trials also indicated severe subfertility. Spermatocytes of cKO mice showed failure to complete prophase I of meiosis with arrest at the midpachytene stage. Staining of synaptonemal complex protein 3 and γH2AX, markers of the meiotic synaptonemal complex and DNA damage, respectively, and super illumination microscopy revealed nonhomologous pairing and synapsis defects. Quantitative RT-PCR showed reduction in the expression of genes critical for prophase I of meiosis, including Spo11 (initiator of meiotic double-stranded breaks), Rec8 (meiotic recombination protein), and Rad21L (RAD21-like, cohesin complex component), as well as those involved in the retinoic acid pathway critical for entry into meiosis. RNA-Seq showed 1036 upregulated and 1638 downregulated genes (false discovery rate <0.05) in the Tardbp cKO testis, impacting meiosis pathways. Our work reveals a crucial role for TDP-43 in male meiosis and suggests that some forms of meiotic arrest seen in infertile men may result from the loss of function of TDP-43.