Effects of 2,4-diaminoquinazoline derivatives on SMN expression and phenotype in a mouse model for spinal muscular atrophy.

Proximal spinal muscular atrophy (SMA), one of the most common genetic causes of infant death, results from the selective loss of motor neurons in the spinal cord. SMA is a consequence of low levels of survival motor neuron (SMN) protein. In humans, the SMN gene is duplicated; SMA results from the loss of SMN1 but SMN2 remains intact. SMA severity is related to the copy number of SMN2. Compounds which increase the expression of SMN2 could, therefore, be potential therapeutics for SMA. Ultrahigh-throughput screening recently identified substituted quinazolines as potent SMN2 inducers. A series of C5-quinazoline derivatives were tested for their ability to increase SMN expression in vivo. Oral administration of three compounds (D152344, D153249 and D156844) to neonatal mice resulted in a dose-dependent increase in Smn promoter activity in the central nervous system. We then examined the effect of these compounds on the progression of disease in SMN lacking exon 7 (SMNDelta7) SMA mice. Oral administration of D156844 significantly increased the mean lifespan of SMNDelta7 SMA mice by approximately 21-30% when given prior to motor neuron loss. In summary, the C5-quinazoline derivative D156844 increases SMN expression in neonatal mouse neural tissues, delays motor neuron loss at PND11 and ameliorates the motor phenotype of SMNDelta7 SMA mice.

Mouse Hyal3 encodes a 45- to 56-kDa glycoprotein whose overexpression increases hyaluronidase 1 activity in cultured cells.

Hyaluronidases are enzymes that mediate the breakdown of hyaluronan (HA), a large polysaccharide abundant in the extracellular matrix of vertebrate tissues. Six genes have been predicted to encode hyaluronidases in humans, but the protein products of only SPAM1, HYAL1, and HYAL2 have been characterized. We have now expressed the mouse Hyal3 gene product, hyaluronidase 3 (Hyal3), in Baby Hamster Kidney (BHK) cells and demonstrated the presence of multiple forms of Hyal3 ranging from approximately 45 to 56 kDa in expression lysates. Complete and partial digestions of the expressed protein with PNGase F showed three N-linked oligosaccharides accounted for all forms of Hyal3 detected in expression lysates. Most of these oligosaccharides were Endo H sensitive, indicating that they were high mannose or hybrid N-linked oligosaccharides. Subcellular fractionation of Hyal3-expressing BHK cells by density gradient centrifugation revealed most Hyal3 in a low-density vesicular population. Low levels of Hyal3 were detected in higher density vesicles, but no colocalization with the late endosomal/lysosomal marker Lamp1 was found by immunofluorescence microscopy. BHK cells stably expressing Hyal3 had increased acid-active hyaluronidase activity, but no such activity was detected when Hyal3 was transfected into Hyaluronidase 1 (Hyal1)-deficient fibroblasts. Overexpression of Hyal3 in BHK cells increased the Hyal1 protein and mRNA levels, suggesting that the increased hyaluronidase activity in these cells was due to Hyal1 rather than Hyal3. The results indicate that Hyal3 overexpressed in cultured cells lacks intrinsic hyaluronidase activity and that Hyal3 may contribute to HA metabolism by augmenting the activity of Hyal1.

V490M, a common mutation in 3-phosphoglycerate dehydrogenase deficiency, causes enzyme deficiency by decreasing the yield of mature enzyme.

A deficiency of 3-phosphoglycerate dehydrogenase (PHGDH) is a disorder of serine biosynthesis identified in children with congenital microcephaly, seizures, and severe psychomotor retardation. We report here the identification of the 1468G-->A (V490M) mutation of this gene in two siblings of an Ashkenazi Jewish family, providing further evidence that the V490M mutation is a common, panethnic cause of this deficiency. Using a novel, DNA-based diagnostic test, the mutation was not detected in 400 non-Jewish controls; one heterozygote was found among 400 persons of Ashkenazi Jewish ethnicity. Extensive biochemical studies were undertaken to characterize the effect of this mutation on enzyme activity, turnover, and stability. The V490M PHGDH yielded less than 35% of the activity observed for the wild-type enzyme when overexpressed by transient transfection or when comparing the endogenous activity in fibroblast cells from the patients with controls. Immunoblotting studies showed a comparable reduction in the level of immunoreactive PHGDH in cells expressing the mutant enzyme. Pulse-chase experiments with metabolically labeled PHGDH indicated that this resulted from an increased rate of degradation of the mutant enzyme following its synthesis. Thermolability analyses of mutant and wild-type enzyme activity revealed no significant differences. While others have proposed that the V490M mutation decreases the V(max) of the enzyme, we conclude that this mutation impairs the folding and/or assembly of PHGDH but has minimal effects on the activity or stability of that portion of the V490M mutant that reaches a mature conformation.