James Pickel

Adult hippocampal neurogenesis buffers stress responses and depressive behaviour

Glucocorticoids are released in response to stressful experiences and serve many beneficial homeostatic functions. However, dysregulation of glucocorticoids is associated with cognitive impairments and depressive illness. In the hippocampus, a brain region densely populated with receptors for stress hormones, stress and glucocorticoids strongly inhibit adult neurogenesis. Decreased neurogenesis has been implicated in the pathogenesis of anxiety and depression, but direct evidence for this role is lacking. Here we show that adult-born hippocampal neurons are required for normal expression of the endocrine and behavioural components of the stress response. Using either transgenic or radiation methods to inhibit adult neurogenesis specifically, we find that glucocorticoid levels are slower to recover after moderate stress and are less suppressed by dexamethasone in neurogenesis-deficient mice than intact mice, consistent with a role for the hippocampus in regulation of the hypothalamic–pituitary–adrenal (HPA) axis. Relative to controls, neurogenesis-deficient mice also showed increased food avoidance in a novel environment after acute stress, increased behavioural despair in the forced swim test, and decreased sucrose preference, a measure of anhedonia. These findings identify a small subset of neurons within the dentate gyrus that are critical for hippocampal negative control of the HPA axis and support a direct role for adult neurogenesis in depressive illness.

Peripheral CB1 cannabinoid receptor blockade improves cardiometabolic risk in mouse models of obesity.

Obesity and its metabolic consequences are a major public health concern worldwide. Obesity is associated with overactivity of the endocannabinoid system, which is involved in the regulation of appetite, lipogenesis, and insulin resistance. Cannabinoid-1 receptor (CB1R) antagonists reduce body weight and improve cardiometabolic abnormalities in experimental and human obesity, but their therapeutic potential is limited by neuropsychiatric side effects. Here we have demonstrated that a CB1R neutral antagonist largely restricted to the periphery does not affect behavioral responses mediated by CB1R in the brains of mice with genetic or diet-induced obesity, but it does cause weight-independent improvements in glucose homeostasis, fatty liver, and plasma lipid profile. These effects were due to blockade of CB1R in peripheral tissues, including the liver, as verified through the use of CB1R-deficient mice with or without transgenic expression of CB1R in the liver. These results suggest that targeting peripheral CB1R has therapeutic potential for alleviating cardiometabolic risk in obese patients.

Hepatic cannabinoid receptor-1 mediates diet-induced insulin resistance via inhibition of insulin signaling and clearance in mice.

BACKGROUND & AIMS Obesity-related insulin resistance contributes to cardiovascular disease. Cannabinoid receptor-1 (CB(1)) blockade improves insulin sensitivity in obese animals and people, suggesting endocannabinoid involvement. We explored the role of hepatic CB(1) in insulin resistance and inhibition of insulin signaling pathways. METHODS Wild-type mice and mice with disruption of CB(1) (CB(1)(-/-) mice) or with hepatocyte-specific deletion or transgenic overexpression of CB(1) were maintained on regular chow or a high-fat diet (HFD) to induce obesity and insulin resistance. Hyperinsulinemic-euglycemic clamp analysis was used to analyze the role of the liver and hepatic CB(1) in HFD-induced insulin resistance. The cellular mechanisms of insulin resistance were analyzed in mouse and human isolated hepatocytes using small interfering or short hairpin RNAs and lentiviral knockdown of gene expression. RESULTS The HFD induced hepatic insulin resistance in wild-type mice, but not in CB(1)(-/-) mice or mice with hepatocyte-specific deletion of CB(1). CB(1)(-/-) mice that overexpressed CB(1) specifically in hepatocytes became hyperinsulinemic as a result of reduced insulin clearance due to down-regulation of the insulin-degrading enzyme. However, they had increased hepatic glucose production due to increased glycogenolysis, indicating hepatic insulin resistance; this was further increased by the HFD. In mice with hepatocytes that express CB(1), the HFD or CB(1) activation induced the endoplasmic reticulum stress response via activation of the Bip-PERK-eIF2α protein translation pathway. In hepatocytes isolated from human or mouse liver, CB(1) activation caused endoplasmic reticulum stress-dependent suppression of insulin-induced phosphorylation of akt-2 via phosphorylation of IRS1 at serine-307 and by inducing the expression of the serine and threonine phosphatase Phlpp1. Expression of CB(1) was up-regulated in samples from patients with nonalcoholic fatty liver disease. CONCLUSIONS Endocannabinoids contribute to diet-induced insulin resistance in mice via hepatic CB(1)-mediated inhibition of insulin signaling and clearance.

CD45‐Positive Blood Cells Give Rise to Uterine Epithelial Cells in Mice

The uterine endometrium is composed of epithelial and stromal cells, which undergo extensive degeneration and regeneration in every estrous cycle, and dramatic changes occur during pregnancy. The high turnover of cells requires a correspondingly high level of cell division by progenitor cells in the uterus, but the character and source of these cells remain obscure. In the present study, using a novel transgenic mouse, we showed that CD45‐positive hematopoietic progenitor cells colonize the uterine epithelium and that in pregnancy more than 80% of the epithelium can derive from these cells. Since we also found green fluorescent protein (GFP)‐positive uterine endothelial cells in long‐term GFP bone marrow‐transplanted mice, we conclude that circulating CD45+ cells play an important role in regenerating the uterine epithelium.

Neurologic, Gastric, and Opthalmologic Pathologies in a Murine Model of Mucolipidosis Type IV

Mucolipidosis type IV (MLIV) is an autosomal recessive lysosomal storage disorder caused by mutations in the MCOLN1 gene, which encodes the 65-kDa protein mucolipin-1. The most common clinical features of patients with MLIV include severe mental retardation, delayed motor milestones, ophthalmologic abnormalities, constitutive achlorhydria, and elevated plasma gastrin levels. Here, we describe the first murine model for MLIV, which accurately replicates the phenotype of patients with MLIV. The Mcoln1(-/-) mice present with numerous dense inclusion bodies in all cell types in brain and particularly in neurons, elevated plasma gastrin, vacuolization in parietal cells, and retinal degeneration. Neurobehavioral assessments, including analysis of gait and clasping, confirm the presence of a neurological defect. Gait deficits progress to complete hind-limb paralysis and death at age ~8 mo. The Mcoln1(-/-) mice are born in Mendelian ratios, and both male and female Mcoln1(-/-) mice are fertile and can breed to produce progeny. The creation of the first murine model for human MLIV provides an excellent system for elucidating disease pathogenesis. In addition, this model provides an invaluable resource for testing treatment strategies and potential therapies aimed at preventing or ameliorating the abnormal lysosomal storage in this devastating neurological disorder.

Neuropathology of the Mcoln1-/-Knockout Mouse Model of Mucolipidosis Type IV

The recently developed Mcoln1−/− knockout mouse provides a novel model for analyzing mucolipin 1 function and mucolipidosis type IV disease. Here we characterize the neuropathology of Mcoln1−/− mouse at the end stage. Evidence of ganglioside accumulation, including increases in GM2, GM3, and GD3 and redistribution of GM1, was found throughout the central nervous system (CNS) independent of significant cholesterol accumulation. Unexpectedly, colocalization studies using immunofluorescence confocal microscopy revealed that GM1 and GM2 were present in separate vesicles within individual neurons. While GM2 was significantly colocalized with LAMP2, consistent with late-endosomal/lysosomal processing, some GM2-immunoreactivity occurred in LAMP2-negative sites, suggesting involvement of other vesicular systems. P62/Sequestosome 1 (P62/SQSTM1) inclusions were also identified in the CNS of the Mcoln1−/− mouse, suggesting deficiencies in protein degradation. Glial cell activation was increased in brain, and there was evidence of reduced myelination in cerebral and cerebellar white matter tracts. Autofluorescent material accumulated throughout the brains of the knockout mice. Finally, axonal spheroids were prevalent in white matter tracts and Purkinje cell axons. This neuropathological characterization of the Mcoln1−/− mouse provides an important step in understanding how mucolipin 1 loss of function affects the CNS and contributes to mucolipidosis type IV disease.

eIF2α phosphorylation-dependent translation in CA1 pyramidal cells impairs hippocampal memory consolidation without affecting general translation

Protein synthesis inhibitor antibiotics are widely used to produce amnesia, and have been recognized to inhibit general or global mRNA translation in the basic translational machinery. For instance, anisomycin interferes with protein synthesis by inhibiting peptidyl transferase or the 80S ribosomal function. Therefore, de novo general or global protein synthesis has been thought to be necessary for long-term memory formation. However, it is unclear which mode of translation—gene-specific translation or general/global translation—is actually crucial for the memory consolidation process in mammalian brains. Here, we generated a conditional transgenic mouse strain in which double-strand RNA-dependent protein kinase (PKR)-mediated phosphorylation of eIF2α, a key translation initiation protein, was specifically increased in hippocampal CA1 pyramidal cells by the chemical inducer AP20187. Administration of AP20187 significantly increased activating transcription factor 4 (ATF4) translation and concomitantly suppressed CREB-dependent pathways in CA1 cells; this led to impaired hippocampal late-phase LTP and memory consolidation, with no obvious reduction in general translation. Conversely, inhibition of general translation by low-dose anisomycin failed to block hippocampal-dependent memory consolidation. Together, these results indicated that CA1-restricted genetic manipulation of particular mRNA translations is sufficient to impair the consolidation and that consolidation of memories in CA1 pyramidal cells through eIF2α dephosphorylation depends more on transcription/translation of particular genes than on overall levels of general translation. The present study sheds light on the critical importance of gene-specific translations for hippocampal memory consolidation.

A regulatory loop involving PAX6, MITF, and WNT signaling controls retinal pigment epithelium development.

The separation of the optic neuroepithelium into future retina and retinal pigment epithelium (RPE) is a critical event in early eye development in vertebrates. Here we show in mice that the transcription factor PAX6, well-known for its retina-promoting activity, also plays a crucial role in early pigment epithelium development. This role is seen, however, only in a background genetically sensitized by mutations in the pigment cell transcription factor MITF. In fact, a reduction in Pax6 gene dose exacerbates the RPE-to-retina transdifferentiation seen in embryos homozygous for an Mitf null allele, and it induces such a transdifferentiation in embryos that are either heterozygous for the Mitf null allele or homozygous for an RPE–specific hypomorphic Mitf allele generated by targeted mutation. Conversely, an increase in Pax6 gene dose interferes with transdifferentiation even in homozygous Mitf null embryos. Gene expression analyses show that, together with MITF or its paralog TFEC, PAX6 suppresses the expression of Fgf15 and Dkk3. Explant culture experiments indicate that a combination of FGF and DKK3 promote retina formation by inhibiting canonical WNT signaling and stimulating the expression of retinogenic genes, including Six6 and Vsx2. Our results demonstrate that in conjunction with Mitf/Tfec Pax6 acts as an anti-retinogenic factor, whereas in conjunction with retinogenic genes it acts as a pro-retinogenic factor. The results suggest that careful manipulation of the Pax6 regulatory circuit may facilitate the generation of retinal and pigment epithelium cells from embryonic or induced pluripotent stem cells.

Gamma motor neurons express distinct genetic markers at birth and require muscle spindle-derived GDNF for postnatal survival

BackgroundGamma motor neurons (γ-MNs) selectively innervate muscle spindle intrafusal fibers and regulate their sensitivity to stretch. They constitute a distinct subpopulation that differs in morphology, physiology and connectivity from α-MNs, which innervate extrafusal muscle fibers and exert force. The mechanisms that control the differentiation of functionally distinct fusimotor neurons are unknown. Progress on this question has been limited by the absence of molecular markers to specifically distinguish and manipulate γ-MNs. Recently, it was reported that early embryonic γ-MN precursors are dependent on GDNF. Using this knowledge we characterized genetic strategies to label developing γ-MNs based on GDNF receptor expression, showed their strict dependence for survival on muscle spindle-derived GDNF and generated an animal model in which γ-MNs are selectively lost.ResultsIn mice heterozygous for both the Hb9::GFP transgene and a tau-lacZ-labeled (TLZ) allele of the GDNF receptor Gfrα1, we demonstrated that small motor neurons with high Gfrα1-TLZ expression and lacking Hb9::GFP display structural and synaptic features of γ-MNs and are selectively lost in mutants lacking target muscle spindles. Loss of muscle spindles also results in the downregulation of Gfrα1 expression in some large diameter MNs, suggesting that spindle-derived factors may also influence populations of α-MNs with β-skeletofusimotor collaterals. These molecular markers can be used to identify γ-MNs from birth to the adult and to distinguish γ- from β-motor axons in the periphery. We also found that postnatal γ-MNs are also distinguished by low expression of the neuronal nuclear protein (NeuN). With these markers of γ-MN identity, we show after conditional elimination of GDNF from muscle spindles that the survival of γ-MNs is selectively dependent on spindle-derived GDNF during the first 2 weeks of postnatal development.ConclusionNeonatal γ-MNs display a unique molecular profile characterized by the differential expression of a series of markers - Gfrα1, Hb9::GFP and NeuN - and the selective dependence on muscle spindle-derived GDNF. Deletion of GDNF expression from muscle spindles results in the selective elimination of γ-MNs with preservation of the spindle and its sensory innervation. This provides a mouse model with which to explore the specific role of γ-fusimotor activity in motor behaviors.

Transgenic mice expressing a cameleon fluorescent Ca2+ indicator in astrocytes and Schwann cells allow study of glial cell Ca2+ signals in situ and in vivo.

Glial cell Ca2+ signals play a key role in glial-neuronal and glial-glial network communication. Numerous studies have thus far utilized cell-permeant and injected Ca2+ indicator dyes to investigate glial Ca2+ signals in vitro and in situ. Genetically encoded fluorescent Ca2+ indicators have emerged as novel probes for investigating cellular Ca2+ signals. We have expressed one such indicator protein, the YC 3.60 cameleon, under the control of the S100beta promoter and directed its expression predominantly in astrocytes and Schwann cells. Expression of YC 3.60 extended into the entire cellular cytoplasmic compartment and the fine terminal processes of protoplasmic astrocytes and Schwann cell Cajal bands. In the brain, all the cells known to express S100beta in the adult or during development, expressed YC 3.60. While expression was most extensive in astrocytes, other glial cell types that express S100beta, such as NG2 and CNP-positive oligodendrocyte progenitor cells (OP cells), microglia, and some of the large motor neurons in the brain stem, also contained YC 3.60 fluorescence. Using a variety of known in situ and in vivo assays, we found that stimuli known to elicit Ca2+ signals in astrocytes caused substantial and rapid Ca2+ signals in the YC 3.60-expressing astrocytes. In addition, forepaw stimulation while imaging astrocytes through a cranial window in the somatosensory cortex in live mice, revealed robust evoked and spontaneous Ca2+ signals. These results, for the first time, show that genetically encoded reporter is capable of recording activity-dependent Ca2+ signals in the astrocyte processes, and networks.