Tyler K. Ulland

Zebrafish are a powerful model for imaging studies of development and neurobiology. However, most studies have focused on developing zebrafish because of the technical and biological challenges of imaging adult stages. These include increased tissue opacity and illumination depth limitations, and difficulty maintaining life support and anesthesia in a 2–4 cm-long fish. There are currently limited tools for intravital imaging of the adult zebrafish brain. The ability to image the brain in the same individual repeatedly without physical damage would facilitate the use of zebrafish to study aging and neurodegenerative disease. We designed and applied a 3D printable device for non-invasive, repeatable multiphoton neural imaging of genetically non-pigmented adult zebrafish across multiple ages and sizes. Animals successfully recovered after multi-hour imaging sessions and could be imaged repeatedly over periods of weeks to months. We show the utility of this approach through imaging the dynamic cells of the neuroimmune system, as well as radial glial cells and oligodendrocytes. This technique could be widely used and beneficial for other cell-scale neuroimaging studies in adult fish.

Type-1 interferons (IFN-1) are a pleiotropic group of cytokines initially known for their antiviral activity, but recent studies highlight a vital role of

Gout arthritis (GA) flares are unexpected bouts of heat, swelling, and redness resulting in excruciating pain caused by monosodium urate (MSU) crystal depo

Clinical observations suggest that obstructive sleep apnea (OSA) and Alzheimer’s disease (AD) pathology may be linked; however, causal mechanisms and relationships are unclear. To investigate the potential interaction between amyloidosis and intermittent hypoxia (IH), a hallmark of OSA, starting at 4 months of age 5XFAD mice were exposed to chronic IH (CIH) consisting of 20 episodes per hour of hypoxia for 12 h/day, daily for 4 months (males) or 6 months (females). CIH did not induce significant changes in amyloid burden or the number of astrocytes in males or females, but there was a slight decrease in the number of microglia observed in the cortex of 5XFAD mice of both sexes. To further explore this effect, we performed bulk RNA sequencing on isolated microglia. In WT mice, the most robust gene changes induced by CIH were identified in male microglia, many of which were pro-inflammatory. In microglia from 5XFAD mice, compared to normoxia (NX), CIH exposure induced comparatively more DEGs in males. Further, in genes that were upregulated by CIH in WT vs 5XFAD mice of both sexes, there was an enrichment of pathways associated with oxidative phosphorylation, aerobic and cellular respiration, and ATP synthesis. These changes indicate that CIH has a more robust effect on the microglial transcriptome in 5XFAD mice than in WT mice, suggesting that the synergy between AD and OSA pathologies may be driven by metabolic changes in the microglial transcriptome. These observations are particularly interesting given the known sex differences in OSA and AD pathology in human disease.

Cell therapy manufacturing of primary T cells often results in heterogeneous cell populations within a final product, with many cells lacking desired of receptor expression or those that have exhausted or other dysfunctional phenotypes. Here, we design a novel cell-intrinsic strategy to genetically reprogram primary human T cells to autonomously detect and eliminate dysfunctional cells. This integrated detection and elimination process, known as directed fratricide, is programmed via nonviral CRISPR genome-editing to eliminate the T cell receptor (TCR) alpha chain ( TRAC gene knockout) and integrate a chimeric antigen receptor (CAR) against the urokinase-type plasminogen activator receptor (uPAR), also known as CD87. Within these cell products, strong T cell stimulation or activation during manufacturing causes a small subset of cells to express uPAR, which subsequently triggers CAR-mediated killing by a separate subset of cells within the product. This fratricide induces proliferation in the desired cells and destroys undesired cells, a process that could be modeled computationally and controlled robustly via supplements to the culture media. The strategy enabled enrichment of anti-uPAR and anti-GD2 CAR T cell products up to ≥99% CAR+/TCR-, favoring a stem cell memory-like phenotype (CD45RA ^high /CD62L ^high ). Understanding growth dynamics among T cell subsets and reprogramming them via CRISPR could accelerate the biomanufacturing of potent cell products without extensive selection methods. Abstract Figure:

Zebrafish are a powerful model for imaging studies of development and neurobiology. However, most studies have focused on developing zebrafish due to technical and biological challenges of imaging adult stages. These include increased tissue opacity and illumination depth limitations, and difficulty maintaining life support and anesthesia in a 2-4cm long fish. There are currently limited tools for intravital imaging of the adult zebrafish brain. The ability to image the brain in the same individual repeatedly without physical damage would allow zebrafish to be better utilized to study aging and neurodegenerative disease. We designed and applied a 3D-printable device for non-invasive, repeatable multiphoton neural imaging of genetically non-pigmented adult zebrafish from 2 months to 19 months old. Animals successfully recovered after multi-hour imaging sessions and can be imaged repeatedly over periods of weeks to years. We show the utility of this approach through imaging the neuroimmune system, revealing that microglia in aged zebrafish have enhanced cellular dynamics. This technique could be widely used and beneficial for other cell-scale neuroimaging studies in the adult fish. Summary Statement: An open-source, 3D printable device that enables non-invasive neuroimaging in adult zebrafish reveals aging-associated changes in neuroimmune cell behavior.

INTRODUCTION: Short-chain fatty acids (SCFAs), produced by gut microbes, influence Alzheimer’s disease (AD) pathology in animals. Less is known about SCFAs and AD in humans. We profiled feces of adults along the AD continuum to investigate gut microbiome and SCFA associations with AD pathology and cognition. METHODS: We measured SCFAs and bacterial abundances in fecal samples from 287 participants in the Wisconsin Alzheimer’s Disease Research Center and Wisconsin Registry for Alzheimer’s Prevention. We performed regressions examining associations between SCFAs or gut microbes and AD pathology and cognition. RESULTS: Fecal propionate, isovalerate, and propionate-producing bacteria are inversely associated with amyloid status. Mediation analysis found that propionate mediates sex-specific associations between SCFAs and CSF biomarkers. SCFA levels are associated with slower cognitive decline. DISCUSSION: These results link SCFAs and propionate-producing bacteria with AD. This may inform efforts to leverage diet and specific bacteria to boost SCFA production and potentially ameliorate AD progression.

Gout arthritis (GA) is an inflammatory disorder characterized by the deposition of monosodium urate (MSU) crystals within synovial joints due to increased urate concentrations in the body. The NLRP3 inflammasome drives a majority of the inflammatory response to MSU crystals; therefore, we hypothesize pharmaceutical agents that attenuate NLRP3 inflammasome activation could be used to treat GA flares. We screened a drug library containing 875 FDA-approved drugs and identified five drugs that reduced NLRP3 inflammasome activation without causing cytotoxic effects in bone marrow-derived macrophages (BMDM). The best performing and therefore leading candidate, verteporfin, used to treat macular degeneration and other eye disorders, reduced Nlrp3- and Caspase-1-dependent IL-1β and IL-18 secretion by BMDM. Additionally, verteporfin-treated mice showed a marked reduction in paw swelling and pro-inflammatory cytokine/chemokine induction, including inflammasome markers (IL-1β and IL-18), in a MSU-induced mouse model of GA flares. Collectively, these data suggest verteporfin is a NLRP3 inflammasome inhibitor that could be repurposed as a treatment for GA.

Alzheimer’s disease (AD) is the most common cause of dementia, leading to substantial personal, economic, and medical costs to patients and society; it is characterized by the build-up of toxic amyloid-beta (Aβ) and hyperphosphorylated tau. It is crucial to the health of the brain that these proteins are processed or drained effectively, but mounting research has shown that in AD pathology there is dysfunction in the ability of the brain to effectively clear pathological Aβ and tau. In this report, we detail the involvement of one important brain drainage pathway and potential site of Aβ clearance, the cribriform plate lymphatics, in 24-month old APP/PS1 mice. We show that cerebrospinal fluid (CSF) efflux is decreased across the cribriform plate area utilizing multiple methods. Moreover, we demonstrate that Aβ aggregates at the cribriform plate – coating surface of olfactory bulbs (OB), olfactory nerve (ON) bundles, and cribriform plate lymphatic endothelial cells (cpLECs). At 24-months, APP/PS1 mice have increased CD45+ cell infiltration and decreased LYVE-1+ vessel area at the cribriform plate, suggesting local inflammation and lymphatic atrophy. Additionally, cpLECs have higher expression of caspase-3 suggesting the decreased LYVE-1 area is due to cellular toxicity resulting in apoptosis. This study demonstrates that the cribriform plate is an important area for further research elucidating its contribution to AD disease pathogenesis.