Centrosomal protein DZIP1 regulates Hedgehog signaling by promoting cytoplasmic retention of transcription factor GLI3 and affecting ciliogenesis.

The primary cilium is required for Hedgehog signaling. So far, all known ciliogenic proteins regulate Hedgehog signaling through their role in ciliogenesis. Here we show that the mouse DZIP1 regulates Hedgehog signaling through two mechanisms. First, DZIP1 interacts with GLI3, a transcriptional regulator for Hedgehog signaling, and prevents GLI3 from entering the nucleus. Second, DZIP1 is required for ciliogenesis. We show that DZIP1 colocalizes and interacts with CEP164, a protein localizing at appendages of the mother centrioles, and IFT88, a component of the intraflagellar transport (IFT) machinery. Functionally, both CEP164 and Ninein appendage proteins fail to localize to ciliary appendages in Dzip1 mutant cells; IFT components are not recruited to the basal body of cilia. Importantly, the accumulation of GLI3 in the nucleus is independent of loss of primary cilia in Dzip1 mutant cells. Therefore, DZIP1 is the first known ciliogenic protein that regulates Hedgehog signaling through a dual mechanism and that biochemically links IFT machinery with Hedgehog pathway components.

The decoupling of Smoothened from Galphai proteins has little effect on Gli3 protein processing and Hedgehog-regulated chick neural tube patterning.

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.

Cholinergic neuronal deficits in CADASIL.

BACKGROUND AND PURPOSE: Previous evidence from MRI and acetylcholinesterase histochemistry suggests cholinergic fibers are affected in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). METHODS: As a measure of cholinergic function, we assessed choline acetyltransferase (ChAT) activities in the frontal and temporal neocortices and the immunocytochemical distribution of ChAT and p75 neurotrophin receptor (P75(NTR)) by in vitro imaging in the nucleus basalis of Meynert of CADASIL subjects. RESULTS: ChAT activities were significantly reduced by 60% to 70% in frontal and temporal cortices of CADASIL cases, as were ChAT and P75(NTR) immunoreactivities in the nucleus basalis. CONCLUSIONS: Our findings suggest cholinergic neuronal impairment in CADASIL and implicate cholinomimetic therapy for subcortical vascular dementias.

CADASIL-causing mutations do not alter Notch3 receptor processing and activation.

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.

Novel presenilin 1 mutation with profound neurofibrillary pathology in an indigenous Southern African family with early-onset Alzheimer's disease.

Genetically determined Alzheimer's disease (AD) is virtually unknown in Africa. We report clinicopathological findings and a presenilin 1 (PS1) mutation associated with early-onset AD in a large Xhosa family from Southern Africa. Twelve individuals spanning four generations were affected, four of whom underwent clinical and psychometric evaluation. Their phenotype was characterized by memory impairment beginning in the early part of the fifth decade, with progressive dementing illness lasting 6-7 years that did not appear to be modified by the presence of an apolipoprotein E (APOE)-epsilon 4 allele. Initial linkage-based analysis using known DNA markers suggested allele cosegregation with a locus on chromosome 14. Direct sequencing of the PS1 gene disclosed a novel I143M (ATT to ATG at nucleotide 677) mutation that lies in a cluster in the second transmembrane domain of the protein. Examination of the proband's brain at autopsy revealed severe AD pathology characterized by neuronal loss, abundant beta amyloid (A beta) neuritic plaques (A beta 42) and neurofibrillary degeneration extending into the brainstem. The phenotype of the I143M mutation was clearly associated with a high degree of neurofibrillary change compared with early-onset sporadic AD cases. Although sporadic cases of AD do exist in African populations, our study confirms the existence of early-onset familial AD among indigenous Southern Africans.