Catabolic Effects of Human PTH (1-34) on Bone: Requirement of Monocyte Chemoattractant Protein-1 in Murine Model of Hyperparathyroidism.

The bone catabolic actions of parathyroid hormone (PTH) are seen in patients with hyperparathyroidism, or with infusion of PTH in rodents. We have previously shown that the chemokine, monocyte chemoattractant protein-1 (MCP-1), is a mediator of PTH's anabolic effects on bone. To determine its role in PTH's catabolic effects, we continuously infused female wild-type (WT) and MCP-1-/- mice with hPTH or vehicle. Microcomputed tomography (µCT) analysis of cortical bone showed that hPTH-infusion induced significant bone loss in WT mice. Further, µCT analysis of trabecular bone revealed that, compared with the vehicle-treated group, the PTH-treated WT mice had reduced trabecular thickness and trabecular number. Notably, MCP-1-/- mice were protected against PTH-induced cortical and trabecular bone loss as well as from increases in serum CTX (C-terminal crosslinking telopeptide of type I collagen) and TRACP-5b (tartrate-resistant acid phosphatase 5b). In vitro, bone marrow macrophages (BMMs) from MCP-1-/- and WT mice were cultured with M-CSF, RANKL and/or MCP-1. BMMs from MCP-1-/- mice showed decreased multinucleated osteoclast formation compared with WT mice. Taken together, our work demonstrates that MCP-1 has a role in PTH's catabolic effects on bone including monocyte and macrophage recruitment, osteoclast formation, bone resorption, and cortical and trabecular bone loss.

Monocyte chemoattractant protein-1 is a mediator of the anabolic action of parathyroid hormone on bone.

Parathyroid hormone (PTH) has a significant role as an anabolic hormone in bone when administered by intermittent injection. Previous microarray studies in our laboratory have shown that the most highly regulated gene, monocyte chemoattractant protein-1 (MCP-1), is rapidly and transiently induced when hPTH(1-34) is injected intermittently in rats. Through further in vivo studies, we found that rats treated with hPTH(1-34) showed a significant increase in serum MCP-1 levels 2 hours after PTH injection compared with basal levels. Using immunohistochemistry, increased MCP-1 expression in osteoblasts and osteocytes is evident after PTH treatment. PTH also increased the number of marrow macrophages. MCP-1 knockout mice injected daily with hPTH(1-34) showed less trabecular bone mineral density and bone volume compared with wild-type mice as measured by peripheral quantitative computed tomography (pQCT) and micro-computed tomography (µCT). Histomorphometric analysis revealed that the increase in osteoclast surface and osteoclast number observed with intermittent PTH treatment in the wild-type mice was completely eliminated in the MCP-1 null mice, as well as much lower numbers of macrophages. Consequently, the lack of osteoclast and macrophage activity in the MCP-1 null mice was paralleled by a reduction in bone formation. We conclude that osteoblast and osteocyte MCP-1 expression is an important mediator for the anabolic effects of PTH on bone.

Amyloid-ß and tau pathology of Alzheimer's disease induced by diabetes in a rabbit animal model.

Alzheimer's disease (AD) is the major age-dependent disease of the brain, but what instigates late-onset AD is not yet clear. Epidemiological, animal model, and cell biology findings suggest links between AD and diabetes. Although AD pathology is accelerated by diabetes in mice engineered to accumulate human-sequence amyloid-ß (Aß) peptides, they do not adequately model non-inherited AD. We investigated AD-type pathology induced solely by diabetes in genetically unmodified rabbits which generate human-sequence Aß peptides. After 15 weeks, alloxan-treated diabetic rabbits with expected high blood glucose showed ~5-fold increase in Aß40/Aß42 in cortex and hippocampus, and significantly, generated Aß-derived assemblies found in human AD. Deposits of these putative pathogenic toxins were detected by Aß/Aß oligomer antibodies in brain parenchyma and surrounding vasculature, also co-localizing with markedly elevated levels of RAGE. Soluble brain extracts showed diabetes-induced buildup of Aß oligomers on dot-blots. Phospho-tau also was clearly elevated, overlapping with ßIII-tubulin along neuronal tracts. Indications of retina involvement in AD led to examination of AD-type pathology in diabetic retinas and showed Aß accumulation in ganglion and inner nuclear cell layers using Aß/oligomer antibodies, and RAGE again was elevated. Our study identifies emergence of AD pathology in brain and retina as a major consequence of diabetes; implicating dysfunctional insulin signaling in late-onset AD, and a potential relationship between Aß-derived neurotoxins and retinal degeneration in aging and diabetes, as well as AD. AD-type pathology demonstrated in genetically unmodified rabbits calls attention to the considerable potential of the model for investigation of AD pathogenesis, diagnostics, and therapeutics.

miR-124, miR-125b, let-7 and vesicle transport proteins in squid lenses in L. pealei.

PURPOSE: Studies over the past several decades identified parallels between neuron and lens fiber cell morphology, development, and physiology. Consistent with this, mammalian lens fiber cells were shown to express a substantial complement of genes that cluster with respect to synaptic vesicle transport and exocytosis. Expression of these genes in these two cell types also appears consistent with similarities described between lens fiber cell lateral protrusions and neuronal dendrites. Recently, we showed vertebrate neurons and lens fiber cells share expression of a core set of factors that form an interlocking regulatory network which has a fundamental role in determining neural cell identity. These included the REST/NRSF transcription factor, neural RNA binding proteins and miR-124. In addition, we identified miR-125 and let-7 in mammalian lenses that have been shown to regulate dendrite formation in neurons. The present study examined expression of miR-124, miR-125, and let-7 as well as genes involved in vesicle transport in lens in the squid Loligo (also referred to as Doryteuthis) pealei. METHODS: Northern blot, RT-PCR, immunoblots, and in situ detection were used to analyze expression in squid and vertebrate tissues. RESULTS: The present study provided evidence that miR-124, miR-125, let-7 and vesicle transport-related proteins are produced in squid lenses. Consistent with these mRNAs and miRNAs in squid lenses, and polyribosomes shown by others, we detected substantial levels of tRNA and rRNA in anuclear squid lenses which do not produce an epithelial cell layer that would be analogous to vertebrate lenses. CONCLUSIONS: Our study provided evidence that miR-124, miR-125, and let-7, as well as proteins involved in vesicle transport linked with synaptic and cargo vesicle transport in vertebrates are also expressed in squid lenses.

Evidence that "brain-specific" FOX-1, FOX-2, and nPTB alternatively spliced isoforms are produced in the lens.

PURPOSE: Alternative RNA splicing is essential in development and more rapid physiological processes that include disease mechanisms. Studies over the last 20 years demonstrated that RNA binding protein families, which mediate the alternative splicing of a large percentage of genes in mammals, contain isoforms with mutually exclusive expression in non-neural and neural progenitor cells vs. post-mitotic neurons, and regulate the comprehensive reprogramming of alternative splicing during neurogenesis. Polypyrimidine tract binding (PTB) proteins and Fox-1 proteins also undergo mutually exclusive alternative splicing in neural and non-neural cells that regulates their tissue-specific expression and splicing activities. Over the past 50 years, striking morphological similarities noted between lens fiber cells and neurons suggested that cell biology processes and gene expression profiles may be shared as well. Here, we examined mouse and rat lenses to determine if alternative splicing of neuronal nPTB and Fox-1/Fox-2 isoforms also occurs in lenses. METHODS: Immunoblot, immunofluorescence, and RT-PCR were used to examine expression and alternative splicing of transcripts in lens and brain. RESULTS: We demonstrated that exon 10 is predominantly included in nPTB transcripts consistent with nPTB protein in lenses, and that alternatively spliced Fox-1/-2 lens transcripts contain exons that have been considered neuron-specific. We identified a 3' alternative Fox-1 exon in lenses that encodes a nuclear localization signal consistent with its protein distribution detected in fiber cells. Neuronal alternative splicing of kinesin KIF1Bß2 has been associated with PTB/nPTB and Fox-2, and we found that two 'neuron-specific' exons are also included in lenses. CONCLUSIONS: The present study provides evidence that alternative neuronal nPTB and Fox-1/Fox-2 isoforms are also produced in lenses. These findings raise questions regarding the extent these factors contribute to a similar reprogramming of alternative splicing during lens differentiation, and the degree that alternative gene transcripts produced during neurogenesis are also expressed in the lens.

HuB/C/D, nPTB, REST4, and miR-124 regulators of neuronal cell identity are also utilized in the lens.

PURPOSE: An interlocking network of transcription factors, RNA binding proteins, and miRNAs globally regulates gene expression and alternative splicing throughout development, and ensures the coordinated mutually exclusive expression of non-neural and neuronal forms of these factors during neurogenesis. Striking similarities between lens fiber cell and neuron cell morphology led us to determine if these factors are also used in the lens. HuR and polypyrimidine tract binding protein (PTB) have been described as 'global regulators' of RNA alternative splicing, stability, and translation in non-neuronal (including ectodermal) tissues examined to date in diverse species, and REST/NRSF (RE-1 Silencing Transcription Factor/Neuron Restrictive Silencing Factor) represses>2,000 neuronal genes in all non-neuronal tissues examined to date, but has not included the lens. During neurogenesis these factors are replaced by what has been considered neuron-specific HuB/C/D, nPTB, and alternatively spliced REST (REST4), which work with miR-124 to activate this battery of genes, comprehensively reprogram neuronal alternative splicing, and maintain their exclusive expression in post-mitotic neurons. METHODS: Immunoprecipitation, western blot, immunofluorescence, and immunohistochemistry were used to determine the expression and distribution of proteins in mouse and rat lenses. Mobility shift assays were used to examine lenses for REST/NRSF DNA binding activity, and RT-PCR, DNA sequencing, and northern blots were used to identify RNA expression and alternative splicing events in lenses from mouse, rat, and goldfish (N. crassa). RESULTS: We demonstrated that REST, HuR, and PTB proteins are expressed predominantly in epithelial cells in mouse and rat lenses, and showed these factors are also replaced by the predominant expression of REST4, HuB/C/D and nPTB in post-mitotic fiber cells, together with miR-124 expression in vertebrate lenses. REST-regulated gene products were found to be restricted to fiber cells where REST is decreased. These findings predicted nPTB- and HuB/C/D-dependent splicing reactions can also occur in lenses, and we showed Neuronal C-src and Type 1 Neurofibromatosis 1 splicing as well as calcitonin gene related peptide (CGRP) and neural cell adhesion molecule (NCAM-180) alternative transcripts in lenses. Transgenic mice with increased HuD in lens also showed increased growth associated protein 43 (GAP43) and Ca++/Calmodulin dependent kinase IIα (CamKIIα) HuD target gene expression in the lens, similar to brain. CONCLUSIONS: The present study provides the first evidence this fundamental set of regulatory factors, previously considered to have a unique role in governing neurogenesis are also used in the lens, and raises questions about the origins of these developmental factors and mechanisms in lens and neuronal cells that also have a basic role in determining the neuronal phenotype.

Increased expression and local accumulation of the prion protein, Alzheimer Aß peptides, superoxide dismutase 1, and nitric oxide synthases 1 & 2 in muscle in a rabbit model of diabetes.

BACKGROUND: Muscle disease associated with different etiologies has been shown to produce localized accumulations of amyloid and oxidative stress-related proteins that are more commonly associated with neurodegeneration in the brain. In this study we examined changes in muscle tissue in a classic model of diabetes and hyperglycemia in rabbits to determine if similar dysregulation of Alzheimer Aß peptides, the prion protein (PrP), and superoxide dismutase 1 (SOD1), as well as nitric oxide synthases is produced in muscle in diabetic animals. This wild-type rabbit model includes systemic physiological expression of human-like Alzheimer precursor proteins and Aß peptides that are considered key in Alzheimer protein studies. RESULTS: Diabetes was produced in rabbits by injection of the toxic glucose analogue alloxan, which selectively enters pancreatic beta cells and irreversibly decreases insulin production, similar to streptozotocin. Quadriceps muscle from rabbits 16 wks after onset of diabetes and hyperglycemia were analyzed with biochemical and in situ methods. Immunoblots of whole muscle protein samples demonstrated increased PrP, SOD1, as well as neuronal and inducible Nitric oxide synthases (NOS1 and NOS2) in diabetic muscle. In contrast, we detected little change in Alzheimer Aß precursor protein expression, or BACE1 and Presenilin 1 levels. However, Aß peptides measured by ELISA increased several fold in diabetic muscle, suggesting a key role for Aß cleavage in muscle similar to Alzheimer neurodegeneration in this diabetes model. Histological changes in diabetic muscle included localized accumulations of PrP, Aß, NOS1 and 2, and SOD1, and evidence of increased central nuclei and cell infiltration. CONCLUSIONS: The present study provides evidence that several classic amyloid and oxidative stress-related disease proteins coordinately increase in overall expression and form localized accumulations in diabetic muscle. The present study highlights the capacity of this wild-type animal model to produce an array of hallmark pathological features that have also been described in other muscle diseases.

Sustained neocortical neurogenesis after neonatal hypoxic/ischemic injury.

OBJECTIVE: Neocortical neurons are sensitive to hypoxic-ischemic (H-I) injuries at term and their demise contributes to neurological disorders. Here we tested the hypothesis that the subventricular zone of the immature brain regenerates neocortical neurons, and that this response is sustained. METHODS: Systemic injections of 5-bromo-2'-deoxyuridine (BrdU) and intraventricular injections of replication-deficient retroviruses were used to label newly born cells, and confocal microscopy after immunofluorescence was used to phenotype the new cells from several days to several months after perinatal H-I in the postnatal day 6 rat. Quantitative polymerase chain reaction was used to evaluate chemoattractants, growth factors, and receptors. RESULTS: Robust production of new neocortical neurons after perinatal H-I occurs. These new neurons are descendants of the subventricular zone, and they colonize the cell-sparse columns produced by the injury to the neocortex. These columns are populated by reactive astrocytes and microglia. Surprisingly, this neuronogenesis is sustained for months. Molecular analyses demonstrated increased neocortical production of insulin-like growth factor-1 and monocyte chemoattractant factor-1 (but statistically insignificant production of erythropoietin, brain-derived neurotrophic factor, glial-derived neurotrophic factor, and transforming growth factor-alpha). INTERPRETATION: The young nervous system has long been known to possess a greater capacity to recover from injury than the adult system. Our data indicate that H-I injury in the neonatal brain initiates an enduring regenerative response from the subventricular zone. These data suggest that additional mechanisms than those previously surmised contribute to the remarkable ability of the immature brain to recover from injury.