Background: All known cilia-related proteins regulate Hedgehog signaling through their role in ciliogenesis. Results: The centrosomal protein DZIP1 interacts with and sequesters GLI3 transcription factor in the cytoplasm and also regulates ciliogenesis. Conclusion: DZIP1 is the first known cilia-related protein that regulates Hedgehog signaling through a dual mechanism. Significance: Understanding how DZIP1 regulates Hedgehog signaling provides new insights into the molecular mechanism of Hedgehog signal transduction.

Several hereditary small vessel diseases (SVDs) of the brain have been reported in recent years. In 1977, Sourander and Wålinder described hereditary multi-infarct dementia (MID) in a Swedish family. In the same year, Stevens and colleagues reported chronic familial vascular encephalopathy in an English family bearing a similar phenotype. These disorders have invariably been suggested to be cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL) but their genetic identities remain unknown. We used molecular, radiological and neuropathological methods to characterize these disorders. Direct DNA sequencing unexpectedly confirmed that affected members of the English family carried the R141C mutation in the NOTCH3 gene diagnostic of CADASIL. However, we did not detect any pathogenic mutations in the entire 8091 bp reading frame of NOTCH3 or find clear evidence for NOTCH3 gene linkage in the Swedish DNA. This was consistent with the lack of hyperintense signals in the anterior temporal pole and external capsule in Swedish subjects upon magnetic resonance imaging. We further found no evidence for granular osmiophilic material in skin biopsy or post-mortem brain samples of affected members in the Swedish family. In addition, there was distinct lack of NOTCH3 N-terminal fragments in the cerebral microvasculature of the Swedish hereditary MID subjects compared to the intense accumulation in the English family afflicted with CADASIL. Several differences in arteriosclerotic changes in both the grey and white matter were also noted between the disorders. The sclerotic index values, density of collagen IV immunoreactivity in the microvasculature and number of perivascular macrophages were greater in the English CADASIL samples compared to those from the Swedish brains. Multiple approaches suggest that the Swedish family with hereditary MID suspected to be CADASIL has a different novel disorder with dissimilar pathological features and belongs to the growing number of genetically uncharacterized familial SVDs.

The Hedgehog (Hh) signal is transmitted by two receptor molecules, Patched (Ptc) and Smoothened (Smo). Ptc suppresses Smo activity, while Hh binds Ptc and alleviates the suppression, which results in activation of Hh targets. Smo is a seven-transmembrane protein with a long carboxyl terminal tail. Vertebrate Smo has been previously shown to be coupled to Galpha(i) proteins, but the biological significance of the coupling in Hh signal transduction is not clear. Here we show that although inhibition of Galpha(i) protein activity appears to significantly reduce Hh pathway activity in Ptc(-/-) mouse embryonic fibroblasts and the NIH3T3-based Shh-light cells, it fails to derepress Shh- or a Smo-agonist-induced inhibition of Gli3 protein processing, a known in vivo indicator of Hh signaling activity. The inhibition of Galpha(i) protein activity also cannot block the Sonic Hedgehog (Shh)-dependent specification of neural progenitor cells in the neural tube. Consistent with these results, overexpression of a constitutively active Galpha(i) protein, Galpha(i2)QL, cannot ectopically specify the neural cell types in the spinal cord, whereas an active Smo, SmoM2, can. Thus, our results indicate that the Smo-induced Galpha(i) activity plays an insignificant role in the regulation of Gli3 processing and Shh-regulated neural tube patterning.

CADASIL is associated with mutations in the Notch3 gene but the causal mechanisms of the disorder remain unclear. We studied effects of widely established mutations on Notch3 receptor processing and ligand-mediated activation in stable lines of HEK293 and SH-SY5Y cells expressing either human wild-type or mutant Notch3 receptor. None of the four mutations (R90C, R133C, C185R and R449C) affected quantities of the full-length, amino-terminal or carboxyl-terminal fragments and did not impair intracellular trafficking in both cell types. The Jagged 1, Jagged 2 and Delta ligand-mediated S2 site cleavage and signal transduction were also observed to be similar in both wild-type and mutants, which exhibited similar rates of degradation of full-length, amino-terminal and carboxyl-terminal fragments. Our results suggest that the arteriopathy in CADASIL is caused by other mechanisms not necessarily involving Notch3 processing and activation.

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