MAB5210 Sigma-AldrichAnti-Phosphacan Antibody, clone 122.2

Traumatic injury to the CNS results in increased expression and deposition of chondroitin sulfate proteoglycans (CSPGs) that are inhibitory to axonal regeneration. Transforming growth factor-β (TGF-β) has been implicated as a major mediator of these changes, but the mechanisms through which TGF-β regulates CSPG expression are not known. Using lentiviral expressed Smad-specific ShRNA we show that TGF-β induction of CSPG expression in astrocytes is Smad-dependent. However, we find a differential dependence of the synthetic machinery on Smad2 and/or Smad3. TGF-β induction of neurocan and xylosyl transferase 1 required both Smad2 and Smad3, whereas induction of phosphacan and chondroitin synthase 1 required Smad2 but not Smad3. Smad3 knockdown selectively reduced induction of chondroitin-4-sulfotransferase 1 and the amount of 4-sulfated CSPGs secreted by astrocytes. Additionally, Smad3 knockdown in astrocytes was more efficacious in promoting neurite outgrowth of neurons cultured on the TGF-β-treated astrocytes. Our data implicate TGF-β Smad3-mediated induction of 4-sulfation as a critical determinant of the permissiveness of astrocyte secreted CSPGs for axonal growth.

We recently described the boundary-like expression pattern of the extracellular matrix molecule tenascin-C (Tnc) in the developing mouse olfactory bulb (OB) (Shay et al., 2008). In the present study, we test the hypothesis that Tnc inhibits olfactory sensory neuron (OSN) axon growth in the developing OB before glomerulogenesis. The period of time before glomerular formation begins, when axons remain restricted to the developing olfactory nerve layer (ONL), is crucial for axon sorting. Here, we show with in vitro analyses that OSN neurite outgrowth is inhibited by Tnc in a dose-dependent manner and that, in stripe assays, axons preferentially avoid Tnc. Using Tnc-null mice, we also show that that glomerular development is delayed in the absence of Tnc. In wild-type mice, OSN axons coalesce into immature or protoglomeruli, which further differentiate and segregate into glomeruli. Glomeruli are first identifiable as discrete structures at birth. In null mice, glomeruli appear immature at birth, remain fused to the ONL, and have a significantly larger diameter compared with wild-type controls. By postnatal day 4, null glomeruli are indistinguishable from controls. Thus, OSN axons appear delayed in their coalescence into glomerular structures. These data correlate with behavioral reports of Tnc-null mice, which are delayed by 24 h in their acquisition of an olfactory behavior (de Chevigny et al., 2006). Collectively, these data demonstrate that Tnc is an inhibitory boundary molecule in the developing OB during a key period of development.

The cerebral cortex may be compressed in hydrocephalus and some experiments suggest that movement of extracellular substances through the cortex is impaired. We hypothesized that the extracellular compartment is reduced in size and that the composition of the extracellular compartment changes in rat brains with kaolin-induced hydrocephalus.We studied neonatal (newborn) onset hydrocephalus for 1 or 3 weeks, juvenile (3 weeks) onset hydrocephalus for 3-4 weeks or 9 months, and young adult (10 weeks) onset hydrocephalus for 2 weeks, after kaolin injection. Freeze substitution electron microscopy was used to measure the size of the extracellular compartment. Western blotting and immunohistochemistry with quantitative image densitometry was used to study the extracellular matrix constituents, phosphacan, neurocan, NG2, decorin, biglycan, and laminin.The extracellular space in cortical layer 1 was reduced significantly from 16.5 to 9.6% in adult rats with 2 weeks duration hydrocephalus. Western blot and immunohistochemistry showed that neurocan increased only in the periventricular white matter following neonatal induction and 3 weeks duration hydrocephalus. The same rats showed mild decorin increases in white matter and around cortical neurons. Juvenile and adult onset hydrocephalus was associated with no significant changes.We conclude that compositional changes in the extracellular compartment are negligible in cerebral cortex of hydrocephalic rats at various ages. Therefore, the functional change related to extracellular fluid flow should be reversible.

The C57BL/6J-Min/+ (Min/+) mouse bears a mutant Apc gene and therefore is an important in vivo model of intestinal tumorigenesis. Min/+ mice develop adenomas that exhibit loss of the wild-type Apc allele (Apc(Min/-)). Previously, we found that histologically normal enterocytes bearing a truncated Apc protein (Apc(Min/+)) migrated more slowly in vivo than enterocytes with either wild-type Apc (Apc(+/+)) or with heterozygous loss of Apc protein (Apc(1638N)). To study this phenotype further, we determined the effect of the Apc(Min) mutation upon cell-cell adhesion by examining the components of the adherens junction (AJ). We observed a reduced association between E-cadherin and beta-catenin in Apc(Min/+) enterocytes. Subcellular fractionation of proteins from Apc(+/+), Apc(Min/+), and Apc(Min/-) intestinal tissues revealed a cytoplasmic localization of intact E-cadherin only in Apc(Min/+), suggesting E-cadherin internalization in these enterocytes. beta-Catenin tyrosine phosphorylation was also increased in Apc(Min/+) enterocytes, consistent with its dissociation from E-cadherin. Furthermore, Apc(Min/+) enterocytes showed a decreased association between beta-catenin and receptor protein-tyrosine phosphatase beta/zeta (RPTPbeta/zeta), and Apc(Min/-) cells demonstrated an association between beta-catenin and receptor protein-tyrosine phosphatase gamma. In contrast to the Apc(Min/+) enterocytes, Apc(Min/-) adenomas displayed increased expression and association of E-cadherin, beta-catenin, and alpha-catenin relative to Apc(+/+) controls. These data show that Apc plays a role in regulating adherens junction structure and function in the intestine. In addition, discovery of these effects in initiated but histologically normal tissue (Apc(Min/+)) defines a pre-adenoma stage of tumorigenesis in the intestinal mucosa.