Bim is elevated in Alzheimer's disease neurons and is required for beta-amyloid-induced neuronal apoptosis.

The molecules that mediate neuron death in Alzheimer's disease (AD) are largely unknown. We report that beta-amyloid (Abeta), a death-promoting peptide implicated in the pathophysiology of AD, induces the proapoptotic protein Bcl-2 interacting mediator of cell death (Bim) in cultured hippocampal and cortical neurons. We further find that Bim is an essential mediator of Abeta-induced neurotoxicity. Our examination of postmortem AD human brains additionally reveals upregulation of Bim in vulnerable entorhinal cortical neurons, but not in cerebellum, a region usually unaffected by AD. Accumulating evidence links inappropriate induction/activation of cell cycle-related proteins to AD, but their roles in the disease have been unclear. We find that the cell cycle molecule cyclin-dependent kinase 4 (cdk4) and its downstream effector B-myb, are required for Abeta-dependent Bim induction and death in cultured neurons. Moreover, neurons that overexpress Bim in AD brains also show elevated levels of the cell cycle-related proteins cdk4 and phospho-Rb. Our observations indicate that Bim is a proapoptotic effector of Abeta and of dysregulated cell cycle proteins in AD and identify both Bim and cell cycle elements as potential therapeutic targets.

The role of microtubule actin cross-linking factor 1 (MACF1) in the Wnt signaling pathway.

MACF1 (microtubule actin cross-linking factor 1) is a multidomain protein that can associate with microfilaments and microtubules. We found that MACF1 was highly expressed in neuronal tissues and the foregut of embryonic day 8.5 (E8.5) embryos and the head fold and primitive streak of E7.5 embryos. MACF1(-/-) mice died at the gastrulation stage and displayed developmental retardation at E7.5 with defects in the formation of the primitive streak, node, and mesoderm. This phenotype was similar to Wnt-3(-/-) and LRP5/6 double-knockout embryos. In the absence of Wnt, MACF1 associated with a complex that contained Axin, beta-catenin, GSK3beta, and APC. Upon Wnt stimulation, MACF1 appeared to be involved in the translocation and subsequent binding of the Axin complex to LRP6 at the cell membrane. Reduction of MACF1 with small interfering RNA decreased the amount of beta-catenin in the nucleus, and led to an inhibition of Wnt-induced TCF/beta-catenin-dependent transcriptional activation. Similar results were obtained with a dominant-negative MACF1 construct that contained the Axin-binding region. Reduction of MACF1 in Wnt-1-expressing P19 cells resulted in decreased T (Brachyury) gene expression, a DNA-binding transcription factor that is a direct target of Wnt/beta-catenin signaling and required for mesoderm formation. These results suggest a new role of MACF1 in the Wnt signaling pathway.

Microtubule actin crosslinking factor 1b: a novel plakin that localizes to the Golgi complex.

MACF1 (microtubule actin crosslinking factor), also called ACF7 (actin crosslinking family 7) is a cytoskeletal linker protein that can associate with both actin filaments and microtubules. We have identified a novel alternatively spliced isoform of MACF1. We named this isoform MACF1b and renamed the original isoform MACF1a. MACF1b is identical to MACF1a, except that it has a region containing plakin (or plectin) repeats in the middle of the molecule. MACF1b is ubiquitously expressed in adult tissues with especially high levels in the lung. We studied the subcellular localization of MACF1b proteins in mammalian cell lines. In two lung cell lines, MACF1b was chiefly localized to the Golgi complex. Upon treatments that disrupt the Golgi complex, MACF1b redistributed into the cytosol, but remained co-localized with the dispersed Golgi ministacks. MACF1b proteins can be detected in the enriched Golgi fraction by western blotting. The domain of MACF1b that targets it to the Golgi was found at the N-terminal part of the region that contains the plakin repeats. Reducing the level of MACF1 proteins by small-interfering RNA resulted in the dispersal of the Golgi complex.

Mutant small heat-shock protein 27 causes axonal Charcot-Marie-Tooth disease and distal hereditary motor neuropathy.

Charcot-Marie-Tooth disease (CMT) is the most common inherited neuromuscular disease and is characterized by considerable clinical and genetic heterogeneity. We previously reported a Russian family with autosomal dominant axonal CMT and assigned the locus underlying the disease (CMT2F; OMIM 606595) to chromosome 7q11-q21 (ref. 2). Here we report a missense mutation in the gene encoding 27-kDa small heat-shock protein B1 (HSPB1, also called HSP27) that segregates in the family with CMT2F. Screening for mutations in HSPB1 in 301 individuals with CMT and 115 individuals with distal hereditary motor neuropathies (distal HMNs) confirmed the previously observed mutation and identified four additional missense mutations. We observed the additional HSPB1 mutations in four families with distal HMN and in one individual with CMT neuropathy. Four mutations are located in the Hsp20-alpha-crystallin domain, and one mutation is in the C-terminal part of the HSP27 protein. Neuronal cells transfected with mutated HSPB1 were less viable than cells expressing the wild-type protein. Cotransfection of neurofilament light chain (NEFL) and mutant HSPB1 resulted in altered neurofilament assembly in cells devoid of cytoplasmic intermediate filaments.

Protein products of human Gas2-related genes on chromosomes 17 and 22 (hGAR17 and hGAR22) associate with both microfilaments and microtubules.

The human Gas2-related gene on chromosome 22 (hGAR22) encodes two alternatively spliced mRNA species. The longer mRNA encodes a protein with a deduced molecular mass of 36.3 kDa (GAR22alpha), whereas the shorter mRNA encodes a larger protein with a deduced molecular mass of 72.6 kDa (GAR22beta). We show that both hGAR22 proteins contain a calponin homology actin-binding domain and a Gas2-related microtubule-binding domain. Using rapid amplification of cDNA ends, we have cloned the mouse orthologue of hGAR22, mGAR22, and found its protein products to be extremely well conserved. We also report the cDNA cloning of a human Gas2-related gene on chromosome 17 (hGAR17). hGAR17 also encodes two protein isoforms. The overall cytoskeletal binding properties of the hGAR17 and hGAR22 proteins are remarkably similar. hGAR17 mRNA expression is limited to skeletal muscle. Although hGAR22 and mGAR22 mRNAs are expressed nearly ubiquitously, mGAR22 protein can only be detected in testis and brain. Furthermore, only the beta isoform is present in these tissues. GAR22beta expression is induced in a variety of cultured cells by growth arrest. The absolute amounts of GAR22beta protein expressed are low. The beta isoforms of hGAR17 and hGAR22 appear to be able to crosslink microtubules and microfilaments in transfected cells. This finding suggests that the physiological functions of these proteins may involve integration of these two components of the cytoskeleton.

Effects of Charcot-Marie-Tooth-linked mutations of the neurofilament light subunit on intermediate filament formation.

Neurofilaments (NFs) are the major intermediate filaments (IFs) of mature neurons. They play important roles in the structure and function of axons. Recently, two mutations in the neurofilament light (NFL) subunit have been identified in families affected by Charcot-Marie-Tooth (CMT) neuropathy type 2. We have characterized the effects of these NFL mutations on the formation of IF networks using a transient transfection system. Both mutations disrupted the self-assembly of human NFL. The Q333P mutant in the rod domain of NFL also disrupted the formation of rat and human NFL/NFM heteropolymers. The phenotypes produced by the P8R mutation in the head domain of NFL were less severe. The P8R mutant NFL co-polymerized with NFM to form bundled filaments and, less often, aggregates. Our results suggest that alterations in the formation of a normal IF network in neurons elicited by these NFL mutations may contribute to the development of Charcot-Marie-Tooth neuropathy.