Thomas van Groen

Disruption of Intraflagellar Transport in Adult Mice Leads to Obesity and Slow-Onset Cystic Kidney Disease

The assembly of primary cilia is dependent on intraflagellar transport (IFT), which mediates the bidirectional movement of proteins between the base and tip of the cilium. In mice, congenic mutations disrupting genes required for IFT (e.g., Tg737 or the IFT kinesin Kif3a) are embryonic lethal, whereas kidney-specific disruption of IFT results in severe, rapidly progressing cystic pathology. Although the function of primary cilia in most tissues is unknown, in the kidney they are mechanosenstive organelles that detect fluid flow through the tubule lumen. The loss of this flow-induced signaling pathway is thought to be a major contributing factor to cyst formation. Recent data also suggest that there is a connection between ciliary dysfunction and obesity as evidenced by the discovery that proteins associated with human obesity syndromes such as Alström and Bardet-Biedl localize to this organelle. To more directly assess the importance of cilia in postnatal life, we utilized conditional alleles of two ciliogenic genes (Tg737 and Kif3a) to systemically induce cilia loss in adults. Surprisingly, the cystic kidney pathology in these mutants is dependent on the time at which cilia loss was induced, suggesting that cyst formation is not simply caused by impaired mechanosensation. In addition to the cystic pathology, the conditional cilia mutant mice become obese, are hyperphagic, and have elevated levels of serum insulin, glucose, and leptin. We further defined where in the body cilia are required for normal energy homeostasis by disrupting cilia on neurons throughout the central nervous system and on pro-opiomelanocortin-expressing cells in the hypothalamus, both of which resulted in obesity. These data establish that neuronal cilia function in a pathway regulating satiety responses.

Connections of the retrosplenial granular b cortex in the rat

Although the retrosplenial granular b cortex (Rgb) is situated in a critical position between the hippocampal formation and the neocortex, surprisingly few studies have examined its connections carefully. The present experiments use both anterograde and retrograde tracing techniques to characterize the connections of Rgb. The main cortical projections from Rgb are to the caudal part of the anterior cingulate cortex, area 18b, retrosplenial granular a cortex (Rga), and postsubiculum, and less dense terminal fields are present in the prelimbic and caudal occipital cortices. The major subcortical projections are to the anterior thalamic nuclei and the rostral pontine nuclei, and very small terminal fields are present in the caudal dorsomedial part of the striatum, the reuniens and reticular nuclei of the thalamus, and the mammillary bodies. Contralaterally, Rgb primarily projects to itself, i.e., homotypically, and more sparsely projects to Rga and postsubiculum. In general, the axons from Rgb terminate ipsilaterally in cortical layers I and III–V and contralaterally in layer V, with a smaller number of terminals in layers I and VI. Thalamic projections from Rgb target the anteroventral and laterodorsal nuclei of the thalamus, with only a few axons terminating in the anterodorsal nucleus, the reticular nucleus, and the nucleus reuniens of the thalamus. Rgb is innervated by the anterior cingulate cortex, precentral agranular cortex, cortical area 18b, dorsal subiculum, and postsubiculum. Subcortical projections to Rgb originate mainly in the claustrum, the horizontal limb of the diagonal band of Broca, and the anterior thalamic nuclei. These data demonstrate that, in the rat, Rgb is a major nodal point for the integration and subsequent distribution of information to and from the hippocampal formation, the midline limbic and visual cortices, and the thalamus. Thus, similarly to the entorhinal cortex, Rgb in the rat is a prominent gateway for information exchange between the hippocampal formation and other limbic areas of the brain. J. Comp. Neurol. 463:249–263, 2003.

The postsubicular cortex in the rat: characterization of the fourth region of the subicular cortex and its connections

The hippocampal formation contributes importantly to many cognitive functions, and therefore has been a focus of intense anatomical and physiological research. Most of this research has focused on the hippocampus proper and the fascia dentata, and much less attention has been given to the subicular cortex, the origin of most extrinsic projections from the hippocampal formation. The present experiments demonstrate that the postsubiculum is a distinct area of the subicular cortex. The major projections to the postsubiculum originate in the hippocampal formation, the cingulate cortex, and the thalamus (primarily from the anterodorsal (AD) nucleus and to a lesser extent from the anteroventral (AV) and lateral dorsal (LD) nuclei). These projections differ from the thalamic projections to presubiculum and parasubiculum. Efferent projections from the postsubiculum terminate in both cortical and subcortical areas. The cortical projections terminate in the subicular and retrosplenial cortices and in the caudal lateral entorhinal and perirhinal cortices. Subcortical projections primarily end in the AD and the LD nuclei of the thalamus. These thalamic projections end in areas that are distinct from those to which the presubiculum and parasubiculum project. For instance, the postsubiculum has a dense terminal field in the AD nucleus, but presubicular axons terminate predominantly in the AV nucleus. The cortical projections also distinguish postsubiculum. All subicular areas project to the entorhinal cortex, but the postsubicular projection ends in the deep layers (i.e. IV-VI), whereas presubiculum projects to layers I and III, and parasubiculum projects to layer II. Postsubiculum projects to retrosplenial granular b cortex and only incidentally to retrosplenial granular a cortex. In contrast presubiculum projects to the retrosplenial granular a cortex but not to the retrosplenial granular b cortex. These differences clearly mark the postsubiculum, the presubiculum, and the parasubiculum as distinct regions within the subicular cortex and suggest that they subserve different roles in the processing and integration of limbic system information.

The connections of presubiculum and parasubiculum in the rat

The present study describes the differences and similarities between the connections of the presubiculum and parasubiculum based on retrograde and anterograde tracing experiments. The results demonstrate that both areas have several similar afferent connections, particularly those from subcortical areas such as the claustrum, diagonal band of Broca, anterior thalamus, nucleus reuniens, locus coeruleus, and raphe nuclei. Both subicular areas also are innervated by axons originating in the ipsilateral and contralateral entorhinal cortex, presubiculum, and parasubiculum. In contrast to these similarities, most axons innervating the presubiculum originate in the lateral dorsal thalamic nucleus, the claustrum, and the contralateral presubiculum. Conversely, the parasubiculum is innervated primarily by axons that originate in area CA1 of the hippocampus, the basolateral nucleus of the amygdala, and the contralateral presubiculum and parasubiculum. The major efferent projection from the presubiculum and parasubiculum courses bilaterally to the medial entorhinal cortex; however, the results of the present study confirm previous suggestions that presubicular axons terminate almost exclusively in layers I and III, whereas parasubicular axons innervate layer II. The presubiculum also projects to the anteroventral and laterodorsal nuclei of the thalamus, and the lateral ventral portion of the medial mammillary nucleus, whereas the parasubiculum projects prominently to the anterodorsal nucleus of the thalamus, the contralateral presubiculum and parasubiculum, and the lateral dorsal segment of the medial mammillary nucleus. Thus despite some similarities, the major connections of presubiculum and parasubiculum are distinct from one another and distinct from the projections of the adjacent subiculum and postsubiculum. These results suggest that the subicular cortex is considerably more complex than previously envisioned and indicate that each segment may subserve a distinct role in the processing of information by the hippocampal formation.

Pulmonary Angiogenesis in a Rat Model of Hepatopulmonary Syndrome

BACKGROUND & AIMS Hepatopulmonary syndrome (HPS), defined as intrapulmonary vasodilation, occurs in 10%-30% of cirrhotics and increases mortality. In a rat model of HPS induced by common bile duct ligation (CBDL), but not thioacetamide (TAA)-induced nonbiliary cirrhosis, lung capillary density increases, monocytes accumulate in the microvasculature, and signaling factors in the angiogenesis pathway (Akt and endothelial nitric oxide synthase [eNOS]) are activated. Pentoxifylline (PTX) directly decreases lung endothelial Akt and eNOS activation, blocks intravascular monocyte accumulation, and improves experimental HPS; we evaluated whether pulmonary angiogenesis develops in this model. METHODS TAA- and PTX-treated animals were evaluated following CBDL. Lung angiogenesis was assessed by quantifying factor VIII-positive microvessels and levels of von Willebrand factor (vWf), vascular endothelial cadherin (VE-cadherin), and proliferating cell nuclear antigen (PCNA). Angiogenic factors including phospho-Akt, phospho-eNOS, vascular endothelial growth factor (VEGF)-A, and phospho-VEGF receptor-2 (p-VEGFR-2) were compared and monocyte accumulation was assessed. RESULTS Following CBDL, but not TAA exposure, rats developed HPS that was temporally correlated with increased numbers of lung microvessel; increased levels of vWf, VE-cadherin and PCNA; and activation of Akt and eNOS. Angiogenesis was accompanied by increased pulmonary VEGF-A and p-VEGFR-2 levels, with VEGF-A staining in accumulated intravascular monocytes and alveolar endothelial cells. Following CBDL, PTX-treated rats had reduced numbers of microvessels, reduced lung monocyte accumulation, downregulation of pulmonary angiogenic factors, and reduced symptoms of HPS. CONCLUSIONS A specific increase in pulmonary angiogenesis occurs as experimental HPS develops, accompanied by activation of VEGF-A-associated angiogenic pathways. PTX decreases the angiogenesis, reduces the symptoms of HPS, and downregulates VEGF-A mediated pathways.

Pyrrolidine Dithiocarbamate Activates Akt and Improves Spatial Learning in APP/PS1 Mice without Affecting β-Amyloid Burden

Pyrrolidine dithiocarbamate (PDTC) is a clinically tolerated inhibitor of nuclear factor-κB (NF-κB), antioxidant and antiinflammatory agent, which provides protection in brain ischemia models. In neonatal hypoxia–ischemia model, PDTC activates Akt and reduces activation of glycogen synthase kinase 3β (GSK-3β). Because chronic inflammation, oxidative stress, and increased GSK-3β activity are features of Alzheimers disease (AD) pathology, we tested whether PDTC reduces brain pathology and improves cognitive function in a transgenic animal model of AD. A 7 month oral treatment with PDTC prevented the decline in cognition in AD mice without altering β-amyloid burden or gliosis. Moreover, marked oxidative stress and activation of NF-κB were not part of the brain pathology. Instead, the phosphorylated form of GSK-3β was decreased in the AD mouse brain, and PDTC treatment increased the phosphorylation of Akt and GSK-3β. Also, PDTC treatment increased the copper concentration in the brain. In addition, PDTC rescued cultured hippocampal neurons from the toxicity of oligomeric Aβ and reduced tau phosphorylation in the hippocampus of AD mice. Finally, astrocytic glutamate transporter GLT-1, known to be regulated by Akt pathway, was decreased in the transgenic AD mice but upregulated back to the wild-type levels by PDTC treatment. Thus, PDTC may improve spatial learning in AD by interfering with Akt–GSK pathway both in neurons and astrocytes. Because PDTC is capable of transferring external Cu2+ into a cell, and, in turn, Cu2+ is able to activate Akt, we hypothesize that PDTC provides the beneficial effect in transgenic AD mice through Cu2+-activated Akt pathway.

Distinct growth hormone receptor signaling modes regulate skeletal muscle development and insulin sensitivity in mice

Skeletal muscle development, nutrient uptake, and nutrient utilization is largely coordinated by growth hormone (GH) and its downstream effectors, in particular, IGF-1. However, it is not clear which effects of GH on skeletal muscle are direct and which are secondary to GH-induced IGF-1 expression. Thus, we generated mice lacking either GH receptor (GHR) or IGF-1 receptor (IGF-1R) specifically in skeletal muscle. Both exhibited impaired skeletal muscle development characterized by reductions in myofiber number and area as well as accompanying deficiencies in functional performance. Defective skeletal muscle development, in both GHR and IGF-1R mutants, was attributable to diminished myoblast fusion and associated with compromised nuclear factor of activated T cells import and activity. Strikingly, mice lacking GHR developed metabolic features that were not observed in the IGF-1R mutants, including marked peripheral adiposity, insulin resistance, and glucose intolerance. Insulin resistance in GHR-deficient myotubes derived from reduced IR protein abundance and increased inhibitory phosphorylation of IRS-1 on Ser 1101. These results identify distinct signaling pathways through which GHR regulates skeletal muscle development and modulates nutrient metabolism.

Neuronal Zinc Exchange with the Blood Vessel Wall Promotes Cerebral Amyloid Angiopathy in an Animal Model of Alzheimer's Disease

Cerebral amyloid angiopathy (CAA) is common in Alzheimers disease (AD) and may contribute to dementia and cerebral hemorrhage. Parenchymal β-amyloid deposition is dependent on the activity of zinc transporter 3 (ZnT3), a neocortical synaptic vesicle membrane protein that causes enrichment of exchangeable Zn2+ in the vesicle, which is externalized on neurotransmission. However, the contribution of zinc to vascular β-amyloid deposition remains unclear. Here, we identify for the first time an exchangeable pool of Zn2+ in the cerebrovascular wall of normal mice. This histochemically reactive Zn2+ is enriched in CAA in a transgenic mouse model of AD (Tg2576), and a dramatic reduction of CAA occurs after targeted disruption of the Znt3 gene in these mice. Also, in Znt3 knock-out mice, the amount of exchangeable Zn2+ [detected by N-(6-methoxy-8-quinolyl)-p-carboxybenzoylsulphonamide (TFL-Zn)] in the perivascular space was significantly decreased in the neocortex but not in peripheral organs. ZnT3 was not detected in the cerebral vessel walls or in blood components of wild-type mice. Thus, synaptic ZnT3 activity may promote CAA by indirectly raising exchangeable Zn2+ concentrations in the perivascular spaces of the brain.

DNA methylation impacts on learning and memory in aging

Learning and memory are two of the fundamental cognitive functions that confer us the ability to accumulate knowledge from our experiences. Although we use these two mental skills continuously, understanding the molecular basis of learning and memory is very challenging. Methylation modification of DNA is an epigenetic mechanism that plays important roles in regulating gene expression, which is one of the key processes underlying the functions of cells including neurons. Interestingly, a genome-wide decline in DNA methylation occurs in the brain during normal aging, which coincides with a functional decline in learning and memory with age. It has been speculated that DNA methylation in neurons might be involved in memory coding. However, direct evidence supporting the role of DNA methylation in memory formation is still under investigation. This particular function of DNA methylation has not drawn wide attention despite several important studies that have provided supportive evidence for the epigenetic control of memory formation. To facilitate further exploration of the epigenetic basis of memory function, we will review existing studies on DNA methylation that are related to the development and function of the nervous system. We will focus on studies illustrating how DNA methylation regulates neural activities and memory formation via the control of gene expression in neurons, and relate these studies to various age-related neurological disorders that affect cognitive functions.

Hippocampal Aβ42 Levels Correlate with Spatial Memory Deficit in APP and PS1 Double Transgenic Mice

We investigated the role of hippocampal amyloid pathology in spatial learning impairment of a new mouse line carrying mutated human amyloid precursor protein (APP) and presenilin-1 (PS1) transgenes. The APP + PS1 mice were tested in spatial navigation in the water maze and in position discrimination in the T-maze at ages of 3-4 and 11-12 months, before and after the appearance of first amyloid plaques. The APP + PS1 mice were impaired in water maze acquisition and retention only at the age of 11-12 months, but performed equally to controls in the T-maze task at both ages. In the impaired older age group, the levels of total Abeta1-42 in the hippocampus of APP + PS1 mice correlated negatively with the retention score. Here we show for the first time that the age-dependent impairment in memory retention in the traditional water maze of APP + PS1 mice correlates with the amount of total Abeta in hippocampus even at a stage when the amyloid deposits cover less than 1% of the hippocampal volume.