Understanding the physiology and pathology of an organ composed of a variety of cell populations depends critically on genome-wide information on each cell type. Here, we report single-cell transcriptome profiling of over 6,800 freshly dispersed anterior pituitary cells from postpubertal male and female rats. Six pituitary-specific cell types were identified based on known marker genes and characterized: folliculostellate cells and hormone-producing corticotrophs, gonadotrophs, thyrotrophs, somatotrophs, and lactotrophs. Also identified were endothelial and blood cells from the pituitary capillary network. The expression of numerous developmental and neuroendocrine marker genes in both folliculostellate and hormone-producing cells supports that they have a common origin. For several genes, the validity of transcriptome analysis was confirmed by qRT-PCR and single cell immunocytochemistry. Folliculostellate cells exhibit impressive transcriptome diversity, indicating their major roles in production of endogenous ligands and detoxification enzymes, and organization of extracellular matrix. Transcriptome profiles of hormone-producing cells also indicate contributions toward those functions, while also clearly demonstrating their endocrine function. This survey highlights many novel genetic markers contributing to pituitary cell type identity, sexual dimorphism, and function, and points to relationships between hormone-producing and folliculostellate cells.

Proper function of lysosomes is particularly important in neurons, as they cannot dilute accumulated toxic molecules and aggregates by cell division. Thus, impairment of lysosomal function plays an important role in neuronal degeneration and in the pathogenesis of numerous neurodegenerative diseases. In this work we analyzed how inhibition and/or loss of the major lysosomal proteases, the cysteine cathepsins B and L (CtsB/L), affects lysosomal function, cholesterol metabolism and degradation of the key Alzheimer's disease (AD) proteins. Here, we show that cysteine CtsB/L, and not the aspartyl cathepsin D (CtsD), represent a major lysosomal protease(s) that control lysosomal function, intracellular cholesterol trafficking and AD-like amyloidogenic features. Intriguingly, accumulation of free cholesterol in late endosomes/lysosomes upon CtsB/L inhibition resembled a phenotype characteristic for the rare neurodegenerative disorder Niemann-Pick type C (NPC). CtsB/L inhibition and not the inhibition of CtsD led to lysosomal impairment assessed by decreased degradation of EGF receptor, enhanced LysoTracker staining and accumulation of several lysosomal proteins LC3II, NPC1 and NPC2. By measuring the levels of NPC1 and ABCA1, the two major cholesterol efflux proteins, we showed that CtsB/L inhibition or genetic depletion caused accumulation of the NPC1 in lysosomes and downregulation of ABCA1 protein levels and its expression. Furthermore, we revealed that CtsB/L are involved in degradation of the key Alzheimer’s proteins: amyloid-β peptides (Aβ) and C-terminal fragments of the amyloid precursor protein (APP) and in degradation of β-secretase (BACE1). Our results imply CtsB/L as major regulators of lysosomal function and demonstrate that CtsB/L may play an important role in intracellular cholesterol trafficking and in degradation of the key AD proteins. Our findings implicate that enhancing the activity or levels of CtsB/L could provide a promising and a common strategy for maintaining lysosomal function and for preventing and/or treating neurodegenerative diseases.

Neurotrophins are established molecular mediators of neuronal plasticity in the adult brain. We analyzed the impact of aging on brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) protein isoforms, their receptors, and on the expression patterns of multiple 5′ exon-specific BDNF transcripts in the rat cortex and hippocampus throughout the life span of the rat (6, 12, 18, and 24 months of age). ProNGF was increased during aging in both structures. Mature NGF gradually decreased in the cortex, and, in 24-month-old animals, it was 30 % lower than that in adult 6-month-old rats. The BDNF expression did not change during aging, while proBDNF accumulated in the hippocampus of aged rats. Hippocampal total BDNF mRNA was lower in 12-month-old animals, mostly as a result of a decrease of BDNF transcripts 1 and 2. In contrast to the regionspecific regulation of specific exon-containing BDNF mRNAs in adult animals, the same BDNF RNA isoforms (containing exons III, IV, or VI) were present in both brain structures of aged animals. Deficits in neurotrophin signaling were supported by the observed decrease in Trk receptor expression which was accompanied by lower levels of the two main downstream effector kinases, pAkt and protein kinase C. The proteolytic processing of p75NTR observed in 12-monthold rats points to an additional regulatory mechanism in early aging. The changes described herein could contribute to reduced brain plasticity underlying the age-dependent decline in cognitive function.

Traumatic brain injury (TBI) is a widespread cause of death and a major source of adult disability. Subsequent pathological events occurring in the brain after TBI, referred to as secondary injury, continue to damage surrounding tissue resulting in substantial neuronal loss. One of the hallmarks of the secondary injury process is microglial activation resulting in increased cytokine production. Notwithstanding that recent studies demonstrated that caloric restriction (CR) lasting several months prior to an acute TBI exhibits neuroprotective properties, understanding how exactly CR influences secondary injury is still unclear. The goal of the present study was to examine whether CR (50% of daily food intake for 3 months) alleviates the effects of secondary injury on neuronal loss following cortical stab injury (CSI). To this end, we examined the effects of CR on the microglial activation, tumor necrosis factor-α (TNF-α) and caspase-3 expression in the ipsilateral (injured) cortex of the adult rats during the recovery period (from 2 to 28 days) after injury. Our results demonstrate that CR prior to CSI suppresses microglial activation, induction of TNF-α and caspase-3, as well as neurodegeneration following injury. These results indicate that CR strongly attenuates the effects of secondary injury, thus suggesting that CR may increase the successful outcome following TBI.