Ami Mariash

The Relationship between Aβ and Memory in the Tg2576 Mouse Model of Alzheimer's Disease

Transgenic mice expressing mutant amyloid precursor proteins (APPs) have provided important new information about the pathogenesis of Alzheimers disease (AD) histopathology. However, the molecular basis of memory loss in these mice is poorly understood. One of the major impediments has been the difficulty of distinguishing between age-dependent and age-independent behavioral changes. To address this issue we studied in parallel two lines of APP transgenic mice expressing comparable levels of mutant and wild-type human APP. This enabled us to identify age-independent behavioral deficits that were not specifically related to mutant APP expression. When mice with age-independent deficits were eliminated, we detected memory loss in transgenic mice expressing mutant APP (Tg2576 mice) starting at ∼6 months, which coincided with the appearance of detergent-insoluble Aβ aggregates (Aβinsol). Genetically accelerating the formation of Aβinsol resulted in an earlier onset of memory decline. A facile interpretation of these results, namely that memory loss and Aβinsol were closely connected, was rejected when we extended our analysis to include older mice. No obvious correspondence between memory and Aβinsol was apparent in a combined group of old and young mice unless the mice were stratified by age, whereupon inverse correlations between memory and Aβinsol became evident. These results suggested that Aβinsol is a surrogate marker for small assemblies of Aβ that disrupt cognition and occur as intermediates during Aβinsol formation, and they are the first descriptive in vivo data supporting their role in impairing memory. These studies also provide a methodological framework within which to investigate these Aβ assemblies in vivo.

Motor dysfunction and gliosis with preserved dopaminergic markers in human α-synuclein A30P transgenic mice

Alpha-synuclein is a major component of Lewy bodies (LBs) in the substantia nigra and cortex in Parkinsons disease (PD) and dementia with Lewy bodies (DLB), and in glial inclusions in multiple systems atrophy (MSA). Mutations in alpha-synuclein have been associated with autosomal dominant forms of PD. We investigated the clinical and neuropathological effects of overexpression of human alpha-synuclein, alpha-synuclein A30P, and alpha-synuclein A53T under the control of the hamster prion protein (PrP) promoter; 5-15x endogenous levels of protein expression were achieved with widespread neuronal, including nigral, transgene expression. High expression of alpha-synuclein A30P in the Tg5093 line was associated with a progressive motor disorder with rigidity, dystonia, gait impairment, and tremor. Histological analysis of this line showed aberrant expression of the protein in cell soma and progressive CNS gliosis, but no discrete Lewy body-like alpha-synuclein inclusions could be identified. Biochemical analysis demonstrated alpha-synuclein fragmentation. Despite strong expression of the transgene in the nigra, there was no specific deterioration of the nigrostriatal dopaminergic system as assessed by quantitation of nigral tyrosine hydroxylase (TH) containing neurons, striatal TH immunoreactivity, dopamine levels, or dopamine receptor number and function. Lower expressing lines had no specific behavioral or histopathological phenotype. Thus, high expression of mutant human alpha-synuclein resulted in a progressive motor and widespread CNS gliotic phenotype independent of dopaminergic dysfunction in the Tg5093 line.

Altered short-term hippocampal synaptic plasticity in mutant α-synuclein transgenic mice

Hippocampal synaptic plasticity was studied in transgenic mice over-expressing human &agr;-synuclein containing the A30P Parkinsons disease mutation. Medial perforant path-dentate granule cell synapses showed enhanced paired-pulse depression (PPD) for short interpulse intervals (< 200 ms), without differences in basal transmission. Extracellular calcium reduction failed to rescue the enhanced PPD. Paired-pulse facilitation in the CA1 region was normal in slices from transgenic mice, but enhanced synaptic depression was revealed upon repetitive stimulation of the Schaffer collaterals. Long-term potentiation in the CA1 field was not impaired in slices from transgenic mice. These results suggest that mutant &agr;-synuclein accumulation impairs short-term changes in synaptic strength when neurotransmitter availability is limited due to enhanced release probability or repetitive synaptic activity.

Increase in GLUT1 in Smooth Muscle Alters Vascular Contractility and Increases Inflammation in Response to Vascular Injury

Objective—The goal of this study was to test the contributing role of increasing glucose uptake in vascular smooth muscle cells (VSMCs) in vascular complications and disease. Methods and Results—A murine genetic model was established in which glucose trasporter 1 (GLUT1), the non–insulin-dependent glucose transporter protein, was overexpressed in smooth muscle using the sm22&agr; promoter. Overexpression of GLUT1 in smooth muscle led to significant increases in glucose uptake (n=3, P<0.0001) as measured using radiolabeled 2-deoxyglucose. Fasting blood glucose, insulin, and nonesterified fatty acids were unchanged. Contractility in aortic ring segments was decreased in sm22&agr;-GLUT1 mice (n=10, P<0.04). In response to vascular injury, sm22&agr;-GLUT1 mice exhibited a proinflammatory phenotype, including a significant increase in the percentage of neutrophils in the lesion (n=4, P<0.04) and an increase in monocyte chemoattractant protein-1 (MCP-1) immunofluorescence. Circulating haptoglobin and glutathione/total glutathione were significantly higher in the sm22&agr;-GLUT1 mice postinjury compared with controls (n=4, P<0.05), suggesting increased flux through the pentose phosphate pathway. sm22&agr;-GLUT1 mice exhibited significant medial hypertrophy following injury that was associated with a significant increase in the percentage of VSMCs in the media staining positive for nuclear phosphoSMAD2/3 (n=4, P<0.003). Conclusion—In summary, these findings suggest that increased glucose uptake in VSMCs impairs vascular contractility and accelerates a proinflammatory, neutrophil-rich lesion in response to injury, as well as medial hypertrophy, which is associated with enhanced transforming growth factor-&bgr; activity.

Loss of redox factor 1 decreases NF-κB activity and increases susceptibility of endothelial cells to apoptosis

Objective— The aim of this project was to test the hypothesis that redox factor 1 (Ref-1) was a critical upstream determinant of NF-&kgr;B–dependent survival signaling pathways in the vessel wall. Methods and Results— Aortas from hemizygous transgenic mice harboring a single allele of Ref-1 exhibited a significant loss in NF-&kgr;B DNA binding activity. The NF-&kgr;B–dependent survival gene A20 was significantly downregulated in aortas of hemizygous Ref-1 mice, whereas IAP-2 was unchanged. Overexpression of A20 rescued cells from tumor necrosis factor (TNF)-induced apoptosis, suggesting that the loss of A20 in Ref-1 hemizygotes may be a rate-determining step in endothelial cell fate. Deletion of the previously defined redox-sensitive or the AP endonuclease domains of Ref-1 significantly decreased NF-&kgr;B transcriptional activation and endothelial cell survival. Furthermore, TNF-induced apoptosis was significantly potentiated in endothelial cells after delivery of Morpholino antisense oligodeoxynucleotides targeted to Ref-1. Loss of the redox-sensitive domain blocked the ability of Ref-1 to reduce p50; however, loss of the endonuclease domain did not effect p50 reduction, suggesting alternative mechanisms of action of Ref-1 on NF-&kgr;B activity. Conclusions— These findings establish a role for Ref-1 as an upstream determinant of NF-&kgr;B and A20-dependent signaling and endothelial survival in the vessel wall.

Matrix metalloproteinase-9 expression in alveolar extraction sockets of Zoledronic acid-treated rats.

PURPOSE The use of nitrogen-containing bisphosphonates (n-bis) is associated with necrosis of the jaws, also known as bisphosphonate-related osteonecrosis of the jaws (BRONJ); however, the pathophysiology is unknown. Matrix metalloproteinase-9 (MMP-9) expression is essential for normal bone healing and is also required for angiogenesis. N-bis alters MMP-9 expression in vitro and in vivo; therefore, we hypothesized that n-bis alters MMP-9 expression during oral wound healing after tooth extraction. MATERIALS AND METHODS A total accumulated dose of 2.25 mg/kg (n = 20) of Zoledronic acid (ZA) Zometa or saline (control, n = 20) was administered to Sprague-Dawley male rats. Next, both groups had maxillary molar teeth extracted. Rats were sacrificed at postoperative day 1, 3, 7, or 21. Western blotting or multiplex ELISA was used to evaluate proteins of interest. Real-time polymerase chain reaction was used to assess the relative quantities of target gene mRNA. MMP-9 enzymatic activity was assessed by zymography. RESULTS The ZA group showed a statistically significant reduction in bone mineralization rate 21 days after tooth extraction compared with the control group (Student t test, P = .005). Moreover, ZA-treated animals showed a statistically significant increase in MMP-9-specific mRNA at postoperative days 3 (P = .003), 7 (P < .0001), and 21 (P < .0001) and protein on postoperative days 3 (P = .005) and 7 (P < .0001). MMP-9 enzymatic activity was also increased in ZA-treated rats compared with control animals (Student t test, P = .014). We also evaluated the extraction sockets for the presence of tissue inhibitor of MMP-1 (TIMP1), which is an inhibitor of MMP-9 enzymatic activity. TIMP1-specific mRNA and protein were not significantly altered by ZA treatment at the times tested (P > .05). Receptor of NF-κB ligand (RANKL) is known to regulate the expression of MMP-9; we therefore assessed the RANKL expression in our experimental oral wound-healing model. The ZA-treated animals had significantly increased RANKL mRNA at postoperative days 3 (P = .02) and 21 (P = .004), while the protein expression was significantly increased at postoperative days 1 (P < .0001), 7 (P = .02), and 21 (P = .03) compared with the control group. CONCLUSIONS ZA reduced bone mineralization within tooth extraction sockets, suggesting aberrant bone healing. ZA increases the amount and enzymatic activity of MMP-9, while apparently not altering the amount of TIMP1 within extraction sockets. RANKL is increased in ZA-treated rats, which suggests that increased MMP-9 expression is due, in part, to augmented RANKL expression.

Heparan sulfate Ndst1 regulates vascular smooth muscle cell proliferation, vessel size and vascular remodeling

Heparan sulfate proteoglycans are abundant molecules in the extracellular matrix and at the cell surface. Heparan sulfate chains are composed of groups of disaccharides whose side chains are modified through a series of enzymatic reactions. Deletion of these enzymes alters heparan sulfate fine structure and leads to changes in cell proliferation and tissue development. The role of heparan sulfate modification has not been explored in the vessel wall. The goal of this study was to test the hypothesis that altering heparan sulfate fine structure would impact vascular smooth muscle cell (VSMC) proliferation, vessel structure, and remodeling in response to injury. A heparan sulfate modifying enzyme, N-deacetylase N-sulfotransferase1 (Ndst1) was deleted in smooth muscle resulting in decreased N- and 2-O sulfation of the heparan sulfate chains. Smooth muscle specific deletion of Ndst1 led to a decrease in proliferating VSMCs and the circumference of the femoral artery in neonatal and adult mice. In response to vascular injury, mice lacking Ndst1 exhibited a significant reduction in lesion formation. Taken together, these data provide new evidence that modification of heparan sulfate fine structure through deletion of Ndst1 is sufficient to decrease VSMC proliferation and alter vascular remodeling.

Cloning, expression and regulation of the human S14 gene

The rat S14 gene has been a useful model to study carbohydrate and triiodothyronine (T3) regulation of hepatic gene expression. To gain insight into the regulation and function of the S14 gene, we isolated the human S14 gene and studied its sequence, tissue specific expression, and transcriptional regulation by glucose and T3. The deduced amino acid sequence of the human S14 protein is 78% identical to that of the rat. Northern blot analysis showed that the S14-mRNA is a single species in human liver and is not present in human brain or HepG2 cells. Transfection studies in primary hepatocytes revealed that transcription of the human S14 gene is regulated by glucose and T3 in a similar manner to that of the rat gene. However, in HepG2 cells, T3 and glucose did not affect the transcription of the human S14 gene. These observations suggest that the S14 gene is highly conserved in mammals and is similarly regulated by carbohydrate and T3 in vivo. More importantly, the function of the human S14 gene may be critical in lipid metabolism in human liver as the rat S14 gene is in rodents.

Accumulation of VEGFR2 in zoledronic acid-treated endothelial cells

Nitrogen-containing bisphosphonates (BIS) are potent inhibitors of bone resorption and are used in the treatment of a number of medical conditions, including multiple myeloma, breast cancer and osteoporosis. Recent experimental evidence demonstrates that BIS also affect endothelial cell functions and angiogenesis; however, the molecular mechanism(s) are unclear. Vascular endothelial growth factor (VEGF) is a potent pro-angiogenic signal for endothelial cells. BIS inhibit VEGF responses in endothelial cells. The VEGF receptor-2 (VEGFR2) is the main signaling receptor for VEGF in endothelial cells. We hypothesized that altered VEGFR2 expression in BIS-treated endothelial cells may account for these attenuated responses to VEGF. The affect of the BIS zoledronic acid (ZOL) was investigated in human umbilical vein endothelial cells using confocal microscopy, Western blotting, real-time PCR and flow cytometry. VEGFR2 accumulated within the ZOL-treated endothelial cells (p=0.0002), though not on the cell surface (p>0.05). ZOL did not induce VEGFR2-specific mRNA (p>0.05). ZOL inhibited endothelial cell chemotaxis towards VEGF (p=0.001). VEGF stimulation significantly reduced the amount of VEGFR2 in the endothelial cells (p=0.01). This response to VEGF was reduced by ZOL (p>0.05). The effects of ZOL on endothelial cell migration, VEGFR2 protein expression and response to VEGF were attenuated by geranylgeranyl pyrophosphate. Two- and one-way ANOVAs with Tukey or Dunnetts multiple comparison adjustments were used. The data suggest that ZOL induces aberrant VEGFR2 accumulation. This is not likely due to the induction of mRNA transcription, but rather to the disruption of the mevalonate pathway.