Increased cerebral matrix metalloprotease-9 activity is associated with compromised recovery in the diabetic db/db mouse following a stroke.

Diabetes is a major risk factor of stroke and is associated with increased frequency of stroke and a poorer prognosis for recovery. In earlier studies we have utilized type 2 diabetic mouse models of stroke and demonstrated that diabetic db/db and ob/ob mice experience larger infarct volumes and impaired recovery associated with greater infiltration of macrophage following hypoxic-ischemic (H/I) insult than their heterozygous non-diabetic db/+ and ob/+ littermates. To obtain a better understanding of the pathogenesis of the impaired recovery, we have investigated the role of matrix metalloproteases and their endogenous inhibitors in the breakdown of the blood-brain barrier (BBB) following H/I. Diabetic db/db mice showed a significant and more rapid increase in matrix metalloprotease (MMP)-9 mRNA, protein and gelatinolytic activity compared with db/+, which resulted in an increased degradation of occludin and collagen IV and subsequently, an increased BBB permeability and greater infiltration of neutrophils into the infarct area. The expression of the MMPs, especially in the db/+ mice, is preceded by an elevated expression of their endogenous tissue inhibitors of metalloproteases (TIMPs) 1, 2, and 3, whereas in the db/db mice, a lower expression of the TIMPs is associated with greater MMP 3 and 9 expression. These results suggest that an imbalance in the MMPs/TIMPs cascade in the diabetic mouse, particularly MMP-9, results in a greater neutrophil invasion, a compromised BBB and consequently a greater insult.

Establishment of cholinergic neuron-like cell lines with differential vulnerability to nitrosative stress.

Cholinergic cell lines were established by fusion of embryonic day 17 wild-type neurons from rat basal forebrain (BF) and upper brainstem (BS) with N18tg neuroblastoma cells. Isolated clones expressed choline acetyltransferase (ChAT) and neuronal nitric oxide synthase (nNOS) activities that were increased upon differentiation with retinoic acid. Clones from the BF expressed high levels of the tyrosine kinase type A (TrkA) receptor expression and activation of the mitogen-activated kinase ERK2 upon treatment with nerve growth factor. Like wild-type cholinergic populations, the six clones studied were variably resistant to nitric oxide (NO) excess from addition of S-nitroso-N-acetyl-D, L-penicillamine (SNAP). Of these, the BS2 clone exhibited resistance like in vivo BS cholinergic neurons, while the MS10 clone mimicked in vivo BF vulnerability. Apoptosis in response to NO excess was preceded by increases in mitochondrial responses bax/bcl-2 ratios, but cytochrome C was not released. Mitochondrial levels of apoptosis initiating factor (AIF) were either unchanged or increased, and only in MS clones was endonuclease G (EndoG) released. Microarray data indicated the existence of endoplasmic reticular (ER) stress and caspase-4 and caspase-12 were involved in the pathway to DNA fragmentation. The array data also indicated a survival role for mdm2, and its blockade rendered vulnerable the brainstem survivor clone BS2. Akt and ERK1/2 pathways were activated in response to NO and their blockade increased DNA fragmentation. Blockade of GSK-3 alpha/beta, a downstream target of Akt, reduced SNAP toxicity and this was more prominent in basal forebrain clones. We have identified two cholinergic cell lines useful for molecular studies of cholinergic vulnerability. We hypothesize that, in cholinergic neurons, control of ER stress signaling may be a major factor in differential vulnerability.

Aging causes partial loss of basal forebrain but no loss of pontine reticular cholinergic neurons.

Cholinergic degeneration occurs in several neurodegenerative diseases. To investigate whether normal aging causes selective neurodegeneration, we compared counts of cholinergic neurons in the medial septum/vertical limb of the diagonal band and pedunculopontine and laterodorsal tegmental nuclei of the brainstem in young and aged Long-Evans rats characterized for their spatial learning ability in the Morris water maze. A subset of aged rats (aged-unimpaired) learned the spatial learning task as young rats, whereas another group (age-impaired) showed poorer learning than young animals. In the medial septum/diagonal band, there was a significant loss (-23%, P < 0.02) of cholinergic neurons in aged-impaired animals compared with young subjects. In the brainstem, there were no significant differences in cholinergic cell number in any group. This selective loss of cholinergic neurons may, in part, account for the cognitive deficits observed in aging and, considering previous findings in this model, may be related to oxidative stress.

Regulation of Epithelial-to-Mesenchymal Transition Using Biomimetic Fibrous Scaffolds.

Epithelial-to-mesenchymal transition (EMT) is a well-studied biological process that takes place during embryogenesis, carcinogenesis, and tissue fibrosis. During EMT, the polarized epithelial cells with a cuboidal architecture adopt an elongated fibroblast-like morphology. This process is accompanied by the expression of many EMT-specific molecular markers. Although the molecular mechanism leading to EMT has been well-established, the effects of matrix topography and microstructure have not been clearly elucidated. Synthetic scaffolds mimicking the meshlike structure of the basement membrane with an average fiber diameter of 0.5 and 5 µm were produced via the electrospinning of poly(ε-caprolactone) (PCL) and were used to test the significance of fiber diameter on EMT. Cell-adhesive peptide motifs were conjugated to the fiber surface to facilitate cell attachment. Madin-Darby Canine Kidney (MDCK) cells grown on these substrates showed distinct phenotypes. On 0.5 µm substrates, cells grew as compact colonies with an epithelial phenotype. On 5 µm scaffolds, cells were more individually dispersed and appeared more fibroblastic. Upon the addition of hepatocyte growth factor (HGF), an EMT inducer, cells grown on the 0.5 µm scaffold underwent pronounced scattering, as evidenced by the alteration of cell morphology, localization of focal adhesion complex, weakening of cell-cell adhesion, and up-regulation of mesenchymal markers. In contrast, HGF did not induce a pronounced scattering of MDCK cells cultured on the 5.0 µm scaffold. Collectively, our results show that the alteration of the fiber diameter of proteins found in the basement membrane may create enough disturbances in epithelial organization and scattering that might have important implications in disease progression.

Aging elevates metabolic gene expression in brain cholinergic neurons.

The basal forebrain (BF) cholinergic system is selectively vulnerable in human brain diseases, while the cholinergic groups in the upper pons of the brainstem (BS) resist neurodegeneration. Cholinergic neurons (200 per region per animal) were laser-microdissected from five young (8 months) and five aged (24 months) F344 rats from the BF and the BS pontine lateral dorsal tegmental/pedunculopontine nuclei (LDTN/PPN) and their expression profiles were obtained. The bioinformatics program SigPathway was used to identify gene groups and pathways that were selectively affected by aging. In the BF cholinergic system, aging most significantly altered genes involved with a variety of metabolic functions. In contrast, BS cholinergic neuronal age effects included gene groupings related to neuronal plasticity and a broad range of normal cellular functions. Transcription factor GA-binding protein alpha (GABPalpha), which controls expression of nuclear genes encoding mitochondrial proteins, was more strongly upregulated in the BF cholinergic neurons (+107%) than in the BS cholinergic population (+40%). The results suggest that aging elicits elevates metabolic activity in cholinergic populations and that this occurs to a much greater degree in the BF group than in the BS group.

Pathway analysis of primary central nervous system lymphoma.

Primary central nervous system (CNS) lymphoma (PCNSL) is a diffuse large B-cell lymphoma (DLBCL) confined to the CNS. A genome-wide gene expression comparison between PCNSL and non-CNS DLBCL was performed, the latter consisting of both nodal and extranodal DLBCL (nDLBCL and enDLBCL), to identify a "CNS signature." Pathway analysis with the program SigPathway revealed that PCNSL is characterized notably by significant differential expression of multiple extracellular matrix (ECM) and adhesion-related pathways. The most significantly up-regulated gene is the ECM-related osteopontin (SPP1). Expression at the protein level of ECM-related SPP1 and CHI3L1 in PCNSL cells was demonstrated by immunohistochemistry. The alterations in gene expression can be interpreted within several biologic contexts with implications for PCNSL, including CNS tropism (ECM and adhesion-related pathways, SPP1, DDR1), B-cell migration (CXCL13, SPP1), activated B-cell subtype (MUM1), lymphoproliferation (SPP1, TCL1A, CHI3L1), aggressive clinical behavior (SPP1, CHI3L1, MUM1), and aggressive metastatic cancer phenotype (SPP1, CHI3L1). The gene expression signature discovered in our study may represent a true "CNS signature" because we contrasted PCNSL with wide-spectrum non-CNS DLBCL on a genomic scale and performed an in-depth bioinformatic analysis.