Summary Blood-borne factors regulate adult hippocampal neurogenesis and cognition in mammals. We report that elevating circulating unacylated-ghrelin (UAG), using both pharmacological and genetic methods, reduced hippocampal neurogenesis and plasticity in mice. Spatial memory impairments observed in ghrelin-O-acyl transferase-null (GOAT −/− ) mice that lack acyl-ghrelin (AG) but have high levels of UAG were rescued by acyl-ghrelin. Acyl-ghrelin-mediated neurogenesis in vitro was dependent on non-cell-autonomous BDNF signaling that was inhibited by UAG. These findings suggest that post-translational acylation of ghrelin is important to neurogenesis and memory in mice. To determine relevance in humans, we analyzed circulating AG:UAG in Parkinson disease (PD) patients diagnosed with dementia (PDD), cognitively intact PD patients, and controls. Notably, plasma AG:UAG was only reduced in PDD. Hippocampal ghrelin-receptor expression remained unchanged; however, GOAT + cell number was reduced in PDD. We identify UAG as a regulator of hippocampal-dependent plasticity and spatial memory and AG:UAG as a putative circulating diagnostic biomarker of dementia.

Mucolipidosis IV (MLIV) is an ultra-rare, recessively inherited lysosomal disorder resulting from inactivating mutations in MCOLN1, the gene encoding the lysosomal cation channel TRPML1. The disease primarily affects the central nervous system (CNS) and manifests in the first year with cognitive and motor developmental delay, followed by a gradual decline in neurological function across the second decade of life, blindness, and premature death in third or fourth decades. Brain pathology manifestations in MLIV are consistent with hypomyelinating leukodystrophy with brain iron accumulation. Presently, there are no approved or investigational therapies for MLIV, and pathogenic mechanisms remain largely unknown. The MLIV mouse model, Mcoln1−/− mice, recapitulates all major manifestations of the human disease. Here, to better understand the pathological mechanisms in the MLIV brain, we performed cell type specific LC–MS/MS proteomics analysis in the MLIV mouse model and reconstituted molecular signatures of the disease in either freshly isolated populations of neurons, astrocytes, oligodendrocytes, and neural stem cells, or whole tissue cortical homogenates from young adult symptomatic Mcoln1−/− mice. Our analysis confirmed on the molecular level major histopathological hallmarks of MLIV universally present in Mcoln1−/− tissue and brain cells, such as hypomyelination, lysosomal dysregulation, and impaired metabolism of lipids and polysaccharides. Importantly, pathway analysis in brain cells revealed mitochondria-related alterations in all Mcoln1−/− brain cells, except oligodendrocytes, that was not possible to resolve in whole tissue. We also report unique proteome signatures and dysregulated pathways for each brain cell population used in this study. These data shed new light on cell-intrinsic mechanisms of MLIV and provide new insights for biomarker discovery and validation to advance translational studies for this disease.

Neuronal mitochondrial fragmentation is a phenotype exhibited in models of neurodegeneration such as Parkinson's disease. Delineating the dysfunction in mitochondrial dynamics found in diseased states can aid our understanding of underlying mechanisms of disease progression and possibly identify novel therapeutic approaches. Advances in microscopy and the availability of intuitive open-access software have accelerated the rate of image acquisition and analysis, respectively. These developments allow routine biology researchers to rapidly turn hypotheses into results. In this protocol, we describe the utilization of cell culture techniques, high-content imaging (HCI), and the subsequent open-source image analysis pipeline for the quantification of mitochondrial fragmentation in the context of a rotenone-based in vitro Parkinson's disease model.

Neuronal mitochondrial fragmentation is a phenotype exhibited in models of neurodegeneration such as Parkinson's Disease. Delineating the dysfunction in mitochondrial dynamics found in diseased states can aid our understanding of underlying mechanisms for disease progression and possibly identify novel therapeutic approaches. Advances in microscopy and the availability of intuitive open-access software has accelerated the rate of image acquisition and analysis, respectively. These developments allow routine biology researchers to rapidly turn hypotheses into results. In this protocol, we describe the utilisation of cell culture techniques, high-content imaging (HCI), and subsequent open-source image analysis pipeline for the quantification of mitochondrial fragmentation in the context of an invitro Parkinson's Disease model. This protocol details wet-lab sample preparation and subsequent image acquisition procedures. The images acquired in this protocol are saved onto a physical or cloud storage platform. A step-by-step guide to build and execute an automated CellProfiler (https://cellprofiler.org/) analysis pipeline for the extraction of mitochondrial measurements will be detailed in 'Basic Protocol 2'.In Basic Protocol 1 we acquire images for subsequent data measurements related to mitochondrial health and fragmentation status. We utilise an in-vitro rotenone-based PD model using which results in increased cytotoxicity, coupled with decreased neuron viability and neuron loss (see Figure 12). At the experimental endpoint, Multitracker Orange (HCS Mitochondrial Health Kit, Invitrogen, Cat. No #H10295) is used to label mitochondria with polarized membranes. In this assay, signal intensity in the orange channel is proportional to membrane potential and mitochondrial health. The experimental set-up was designed for testing the effect of a range of putative anti-PD compounds on mitochondria structure and function.