pH dependence of amylin fibrillization.

In type 2 diabetics, the hormone amylin misfolds into amyloid plaques implicated in the destruction of the pancreatic ß-cells that make insulin and amylin. The aggregative misfolding of amylin is pH-dependent, and exposure of the hormone to acidic and basic environments could be physiologically important. Amylin has two ionizable residues between pH 3 and 9: the α-amino group and His18. Our approach to measuring the pKa values for these sites has been to look at the pH dependence of fibrillization in amylin variants that have only one of the two groups. The α-amino group at the unstructured N-terminus of amylin has a pKa near 8.0, similar to the value in random coil models. By contrast, His18, which is involved in the intermolecular ß-sheet structure of the fibrils, has a pKa that is lowered to 5.0 in the fibrils compared to the random coil value of 6.5. The lowered pKa of His18 is due to the hydrophobic environment of the residue, and electrostatic repulsion between positively charged His18 residues on neighboring amylin molecules in the fibril. His18 acts as an electrostatic switch inhibiting fibrillization in its charged state. The presence of a charged side chain at position 18 also affects fibril morphology and lowers amylin cytotoxicity toward a MIN6 mouse model of pancreatic ß-cells. In addition to the two expected pKa values, we detected an apparent pKa of ~4.0 for the amylin-derived peptide NAc-SNNFGAILSS-NH2, which has no titratable groups. This pKa is due to the pH-induced ionization of the dye thioflavin T. By using alternative methods to follow fibrillization such as the dye Nile Red or turbidimetry, we were able to distinguish between the titration of the dye and groups on the peptide. Large differences in reaction kinetics were observed between the different methods at acidic pH, because of charges on the ThT dye, which hinder fibril formation much like the charges on the protein.

Midbrain dopamine neurons associated with reward processing innervate the neurogenic subventricular zone.

Coordinated regulation of the adult neurogenic subventricular zone (SVZ) is accomplished by a myriad of intrinsic and extrinsic factors. The neurotransmitter dopamine is one regulatory molecule implicated in SVZ function. Nigrostriatal and ventral tegmental area (VTA) midbrain dopamine neurons innervate regions adjacent to the SVZ, and dopamine synapses are found on SVZ cells. Cell division within the SVZ is decreased in humans with Parkinson's disease and in animal models of Parkinson's disease following exposure to toxins that selectively remove nigrostriatal neurons, suggesting that dopamine is critical for SVZ function and nigrostriatal neurons are the main suppliers of SVZ dopamine. However, when we examined the aphakia mouse, which is deficient in nigrostriatal neurons, we found no detrimental effect to SVZ proliferation or organization. Instead, dopamine innervation of the SVZ tracked to neurons at the ventrolateral boundary of the VTA. This same dopaminergic neuron population also innervated the SVZ of control mice. Characterization of these neurons revealed expression of proteins indicative of VTA neurons. Furthermore, exposure to the neurotoxin MPTP depleted neurons in the ventrolateral VTA and resulted in decreased SVZ proliferation. Together, these results reveal that dopamine signaling in the SVZ originates from a population of midbrain neurons more typically associated with motivational and reward processing.

Subventricular zone-mediated ependyma repair in the adult mammalian brain.

The subventricular zone (SVZ) of the adult mouse brain is a narrow stem cell niche that lies along the length of the lateral wall of the lateral ventricles. The SVZ supports neurogenesis throughout adulthood; however, with increasing age, the ventral SVZ deteriorates and only the dorsolateral SVZ remains neurogenic. Associated with the elderly dorsolateral SVZ, we reported previously an increased number of astrocytes interposed within the adjacent ependymal lining. Here, we show that astrocytes integrated within the ependyma are dividing, BrdU-labeled astrocytes that share cellular adherens with neighboring ependymal cells. By tracking BrdU-labeled astrocytes over time, we observed that, as they incorporated within the ependyma, they took on antigenic and morphologic characteristics of ependymal cells, suggesting a novel form of SVZ-supported "regenerative" repair in the aging brain. A similar form of SVZ-mediated ependyma repair was also observed in young mice after mild ependymal cell denudation with low dosages of neuraminidase. Together, this work identifies a novel non-neuronal mechanism of regenerative repair by the adult SVZ.

Neuroblast protuberances in the subventricular zone of the regenerative MRL/MpJ mouse.

The MRL mouse is unique in its capacity for regenerative healing of wounds. This regenerative ability includes complete closure, with little scarring, of wounds to the ear pinna and repair of cardiac muscle, without fibrosis, following cryoinjury. Here, we examine whether neurogenic zones within the MRL brain show enhanced regenerative capacity. The largest neurogenic zone in the adult brain, the subventricular zone (SVZ), lies adjacent to the lateral wall of the lateral ventricle and is responsible for replacement of interneuron populations within the olfactory bulb. Initial gross observation of the anterior forebrain in MRL mice revealed enlarged lateral ventricles; however, little neurodegeneration was detected within the SVZ or surrounding tissues. Instead, increased proliferation within the SVZ was observed, based on incorporation of the thymidine analogue bromodeoxyuridine. Closer examination using electron microscopy revealed that a significant number of SVZ astrocytes interpolated within the ependyma and established contact with the ventricle. In addition, subependymal, protuberant nests of cells, consisting primarily of neuroblasts, were found along the anterior SVZ of MRL mice. Whole mounts of the lateral wall of the lateral ventricle stained for the neuroblast marker doublecortin revealed normal formation of chains of migratory neuroblasts along the entire wall and introduction of enhanced green fluorescent protein-tagged retrovirus into the lateral ventricles confirmed that newly generated neuroblasts were able to track into the olfactory bulb.

The aging neurogenic subventricular zone.

In the adult mouse brain, the subventricular zone (SVZ) is a neurogenic stem cell niche only 4-5 cell diameters thick. Within this narrow zone, a unique microenvironment supports stem cell self-renewal, gliogenesis or neurogenesis lineage decisions and tangential migration of newly generated neurons out of the SVZ and into the olfactory bulb. However, with aging, SVZ neurogenesis declines. Here, we examine the dynamic interplay between SVZ cytoarchitecture and neurogenesis through aging. Assembly of high-resolution electron microscopy images of corresponding coronal sections from 2-, 10- and 22-month-old mice into photomontages reveal a thinning of the SVZ with age. Following a 2-h BrdU pulse, we detect a significant decrease in cell proliferation from 2 to 22 months. Neuroblast numbers decrease with age, as do transitory amplifying progenitor cells, while both SVZ astrocytes and adjacent ependymal cells remain relatively constant. At 22 months, only residual pockets of neurogenesis remain and neuroblasts become restricted to the anterior dorsolateral horn of the SVZ. Within this dorsolateral zone many key components of the younger neurogenic niche are maintained; however, in the aged SVZ, increased numbers of SVZ astrocytes are found interposed within the ependyma. These astrocytes co-label with markers to ependymal cells and astrocytes, form intercellular adherens junctions with neighboring ependymal cells, and some possess multiple basal bodies of cilia within their cytoplasm. Together, these data reveal an age-related, progressive restriction of SVZ neurogenesis to the dorsolateral aspect of the lateral ventricle with increased numbers of SVZ astrocytes interpolated within the ependyma.

NG2 glial cells provide a favorable substrate for growing axons.

NG2 cells (polydendrocytes) comprise an abundant glial population that is widely and uniformly distributed throughout the developing and mature CNS and are identified by the expression of the NG2 proteoglycan at the cell surface. Although recent electrophysiological studies suggest that they are capable of receiving signals from axon terminals, other studies, based on the finding that the NG2 molecule itself induces growth cone collapse, have led to a widely held speculation that NG2 cells themselves also repel and inhibit growing axons. In this study, we have examined the effects of rat NG2 cells on growing hippocampal and neocortical axons in vitro and in vivo. NG2 cells did not repel growing axons but promoted their growth in vitro, and axonal growth cones formed extensive contacts with NG2 cells both in vitro and in the developing corpus callosum. Punctate immunoreactivity for fibronectin and laminin was found to be colocalized with NG2 on the surface of NG2 cells. Altering the level of cell surface NG2 expression had no effect on the growth-promoting effects of NG2 cells on growing axons. Thus, our study indicates that NG2 cells are not inhibitory to growing axons but provide an adhesive substrate for axonal growth cones and promote their growth even in the presence of elevated levels of the NG2 proteoglycan. These findings suggest a novel role for NG2 cells in facilitating axonal growth during development and regeneration.