Harmine and exendin-4 combination therapy safely expands human ß cell mass in vivo in a mouse xenograft system.

Five hundred thirty-seven million people globally suffer from diabetes. Insulin-producing ß cells are reduced in number in most people with diabetes, but most individuals still have some residual ß cells. However, none of the many diabetes drugs in common use increases human ß cell numbers. Recently, small molecules that inhibit dual tyrosine-regulated kinase 1A (DYRK1A) have been shown to induce immunohistochemical markers of human ß cell replication, and this is enhanced by drugs that stimulate the glucagon-like peptide 1 (GLP1) receptor (GLP1R) on ß cells. However, it remains to be demonstrated whether these immunohistochemical findings translate into an actual increase in human ß cell numbers in vivo. It is also unknown whether DYRK1A inhibitors together with GLP1R agonists (GLP1RAs) affect human ß cell survival. Here, using an optimized immunolabeling-enabled three-dimensional imaging of solvent-cleared organs (iDISCO+) protocol in mouse kidneys bearing human islet grafts, we demonstrate that combination of a DYRK1A inhibitor with exendin-4 increases actual human ß cell mass in vivo by a mean of four- to sevenfold in diabetic and nondiabetic mice over 3 months and reverses diabetes, without alteration in human α cell mass. The augmentation in human ß cell mass occurred through mechanisms that included enhanced human ß cell proliferation, function, and survival. The increase in human ß cell survival was mediated, in part, by the islet prohormone VGF. Together, these findings demonstrate the therapeutic potential and favorable preclinical safety profile of the DYRK1A inhibitor-GLP1RA combination for diabetes treatment.

Development and cadherin-mediated control of prefrontal corticostriatal projections in mice.

Action-outcome associations depend on prefrontal cortex (PFC) projections to the dorsal striatum. To assess how these projections form, we measured PFC axon patterning, synapse formation, and functional maturation in the postnatally developing mouse striatum. Using Hotspot analysis, we show that PFC axons form an adult-like pattern of clustered terminations in the first postnatal week that remains largely stable thereafter. PFC-striatal synaptic strength is adult-like by P21, while excitatory synapse density increases until adulthood. We then tested how the targeted deletion of a candidate adhesion/guidance protein, Cadherin-8 (Cdh8), from corticostriatal neurons regulates pathway development. Mutant mice showed diminished PFC axon targeting and reduced spontaneous glutamatergic synaptic activity in the dorsal striatum. They also exhibited impaired behavioral performance in action-outcome learning. The data show that PFC-striatal axons form striatal territories through an early, directed growth model and they highlight essential contributions of Cdh8 to the anatomical and functional features critical for the formation of action-outcome associations.

Development of prefrontal corticostriatal connectivity in mice.

Rational decision making is grounded in learning to associate actions with outcomes, a process that depends on projections from prefrontal cortex to dorsomedial striatum. Symptoms associated with a variety of human pathological conditions ranging from schizophrenia and autism to Huntington's and Parkinson's disease point toward functional deficits in this projection, but its development is not well understood, making it difficult to investigate how perturbations in development of this circuitry could contribute to pathophysiology. We applied a novel strategy based on Hotspot Analysis to assess the developmental progression of anatomical positioning of prefrontal cortex to striatal projections. Corticostriatal axonal territories established at P7 expand in concert with striatal growth but remain largely unchanged in positioning through adulthood, indicating they are generated by directed, targeted growth and not modified extensively by postnatal experience. Consistent with these findings, corticostriatal synaptogenesis increased steadily from P7 to P56, with no evidence for widescale pruning. As corticostriatal synapse density increased over late postnatal ages, the strength of evoked PFC input onto dorsomedial striatal projection neurons also increased, but spontaneous glutamatergic synaptic activity was stable. Based on its pattern of expression, we asked whether the adhesion protein, Cdh8, influenced this progression. In mice lacking Cdh8 in PFC corticostriatal projection neurons, axon terminal fields in dorsal striatum shifted ventrally. Corticostriatal synaptogenesis was unimpeded, but spontaneous EPSC frequency declined and mice failed to learn to associate an action with an outcome. Collectively these findings show that corticostriatal axons grow to their target zone and are restrained from an early age, do not undergo postnatal synapse pruning as the most dominant models predict, and that a relatively modest shift in terminal arbor positioning and synapse function has an outsized, negative impact on corticostriatal-dependent behavior.

Protocol to identify and analyze mouse and human quiescent hematopoietic stem cells using flow cytometry combined with confocal imaging.

Mitochondrial membrane potential (MMP) segregates functionally distinct subsets within highly purified hematopoietic stem cells (HSCs). Here, we detail a protocol for FACS isolation of MMP sub-fractions of phenotypically defined mouse and human HSCs. These steps are followed by high-/super-resolution immunofluorescence microscopy of HSCs' lysosomes. While the protocol describes the isolation of quiescent HSCs, which are the most potent subsets, it could also be applied to other HSC subsets. This protocol overcomes some experimental challenges associated with low HSC numbers. For complete details on the use and execution of this protocol, please refer to Liang et al. (2020) and Qiu et al. (2021).

Optical Clearing and 3D Analysis Optimized for Mouse and Human Pancreata.

The pancreas is a heavily innervated organ, but pancreatic innervation can be challenging to comprehensively assess using conventional histological methods. However, recent advances in whole-mount tissue clearing and 3D rendering techniques have allowed detailed reconstructions of pancreatic innervation. Optical clearing is used to enhance tissue transparency and reduce light scattering, thus eliminating the need to section the tissue. Here, we describe a modified version of the optical tissue clearing protocol iDISCO+ (immunolabeling-enabled three-dimensional imaging of solvent-cleared organs) optimized for pancreatic innervation and endocrine markers. The protocol takes 13-19 days, depending on tissue size. In addition, we include protocols for imaging using light sheet and confocal microscopes and for 3D segmentation of pancreatic innervation and endocrine cells using Imaris.

Restraining Lysosomal Activity Preserves Hematopoietic Stem Cell Quiescence and Potency.

Quiescence is a fundamental property that maintains hematopoietic stem cell (HSC) potency throughout life. Quiescent HSCs are thought to rely on glycolysis for their energy, but the overall metabolic properties of HSCs remain elusive. Using combined approaches, including single-cell RNA sequencing (RNA-seq), we show that mitochondrial membrane potential (MMP) distinguishes quiescent from cycling-primed HSCs. We found that primed, but not quiescent, HSCs relied readily on glycolysis. Notably, in vivo inhibition of glycolysis enhanced the competitive repopulation ability of primed HSCs. We further show that HSC quiescence is maintained by an abundance of large lysosomes. Repression of lysosomal activation in HSCs led to further enlargement of lysosomes while suppressing glucose uptake. This also induced increased lysosomal sequestration of mitochondria and enhanced the competitive repopulation ability of primed HSCs by over 90-fold in vivo. These findings show that restraining lysosomal activity preserves HSC quiescence and potency and may be therapeutically relevant.

A 3D atlas of the dynamic and regional variation of pancreatic innervation in diabetes.

Understanding the detailed anatomy of the endocrine pancreas, its innervation, and the remodeling that occurs in diabetes can provide new insights into metabolic disease. Using tissue clearing and whole-organ imaging, we identified the 3D associations between islets and innervation. This technique provided detailed quantification of α and ß cell volumes and pancreatic nerve fibers, their distribution and heterogeneity in healthy tissue, canonical mouse models of diabetes, and samples from normal and diabetic human pancreata. Innervation was highly enriched in the mouse endocrine pancreas, with regional differences. Islet nerve density was increased in nonobese diabetic mice, in mice treated with streptozotocin, and in pancreata of human donors with type 2 diabetes. Nerve contacts with ß cells were preserved in diabetic mice and humans. In summary, our whole-organ assessment allows comprehensive examination of islet characteristics and their innervation and reveals dynamic regulation of islet innervation in diabetes.