Mitochondria exist as a dynamic tubular network with projections that move, break, and reseal in response to local environmental changes. We present evidence that a human dynamin-related protein (Drp1) is specifically required to establish this morphology. Drp1 is a GTPase with a domain structure similar to that of other dynamin family members. To identify the function of Drp1, we transiently transfected cells with mutant Drp1. A mutation in the GTPase domain caused profound alterations in mitochondrial morphology. The tubular projections normally present in wild-type cells were retracted into large perinuclear aggregates in cells expressing mutant Drp1. The morphology of other organelles was unaffected by mutant Drp1. There was also no effect of mutant Drp1 on the transport functions of the secretory and endocytic pathways. By EM, the mitochondrial aggregates found in cells that were transfected with mutant Drp1 appear as clusters of tubules rather than a large mass of coalescing membrane. We propose that Drp1 is important for distributing mitochondrial tubules throughout the cell. The function of this new dynamin-related protein in organelle morphology represents a novel role for a member of the dynamin family of proteins.

Although intradendritic protein synthesis has been documented in adult neurons, the question of whether axons actively synthesize proteins remains controversial. Adult sensory neurons that are conditioned by axonal crush can rapidly extend processes in vitro by regulating the translation of existing mRNAs (Twiss et al., 2000). These regenerating processes contain axonal but not dendritic proteins. Here we show that these axonal processes of adult sensory neurons cultured after conditioning injury contain ribosomal proteins, translational initiation factors, and rRNA. Pure preparations of regenerating axons separated from the DRG cell bodies can actively synthesize proteins in vitro and contain ribosome-bound beta-actin and neurofilament mRNAs. Blocking protein synthesis in these regenerating sensory axons causes a rapid retraction of their growth cones when communication with the cell body is blocked by axotomy or colchicine treatment. These findings indicate that axons of adult mammalian neurons can synthesize proteins and suggest that, under some circumstances, intra-axonal translation contributes to structural integrity of the growth cone in regenerating axons. By immunofluorescence, translation factors, ribosomal proteins, and rRNA were also detected in motor axons of ventral spinal roots analyzed after 7 d in vivo after a peripheral axonal crush injury. Thus, adult motor neurons are also likely capable of intra-axonal protein synthesis in vivo after axonal injury.

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The islet in type 2 diabetes mellitus (T2DM) is characterized by a deficit in ␤ cells and islet amyloid derived from islet amyloid polypeptide (IAPP), a protein coexpressed with insulin by ␤ cells. It is increasingly appreciated that the toxic form of amyloidogenic proteins is not amyloid but smaller membrane-permeant oligomers. Using an antibody specific for toxic oligomers and cryo-immunogold labeling in human IAPP transgenic mice, human insulinoma and pancreas from humans with and without T2DM, we sought to establish the abundance and sites of formation of IAPP toxic oligomers. We conclude that IAPP toxic oligomers are formed intracellularly within the secretory pathway in T2DM. Most striking , IAPP toxic oligomers appear to disrupt membranes of the secretory pathway , and then when adjacent to mitochondria , disrupt mitochondrial membranes. Toxic oligomer-induced secretory pathway and mitochondrial membrane disruption is a novel mechanism to account for cellular dysfunction and apoptosis in T2DM. Type 2 diabetes (T2DM) is characterized by a progressive deficit in ␤ cell function and mass with increased ␤ cell apoptosis.1,2 In common with several neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease, the loss of ␤ cells in T2DM is associated with accumulation of locally expressed misfolded proteins that share a propensity to form amyloid.3 Islet amyloid in T2DM is composed primarily of a 37-amino acid protein, islet amyloid polypeptide (IAPP).3 IAPP is co-expressed and secreted with insulin by pancreatic ␤ cells, and is thought to play a paracrine inhibitory role in regulation of insulin secretion. 4,5 The property of IAPP to form amyloid fibrils depends on IAPP 20-29 . This sequence is closely homologous in humans, nonhuman primates and cats, 6 all of which spontaneously develop T2DM characterized by a deficit in ␤ cell mass and islet amyloid. In contrast, rodent IAPP (mouse and rat) does not have the propensity to form amyloid fibrils due to proline substitutions in IAPP 20-29 and wild-type mice and rats do not spontaneously develop T2DM.There is accumulating evidence that the toxic form of amyloidogenic protein aggregates is distinct from amyloid fibrils. The latter tend to accumulate extracellularly where they are relatively inert.3,7 Abnormal non-fibrillar intracellular IAPP aggregates were noted in human insulinoma tissue adjacent to disrupted intracellular membranes.

Clathrin-coated vesicles mediate diverse processes such as nutrient uptake, downregulation of hormone receptors, formation of synaptic vesicles, virus entry, and transport of biosynthetic proteins to lysosomes. Cycles of coat assembly and disassembly are integral features of clathrin-mediated vesicular transport (Fig. 1a). Coat assembly involves recruitment of clathrin triskelia, adaptor complexes and other factors that influence coat assembly, cargo sequestration, membrane invagination and scission (Fig. 1a). Coat disassembly is thought to be essential for fusion of vesicles with target membranes and for recycling components of clathrin coats to the cytoplasm for further rounds of vesicle formation. In vitro, cytosolic heat-shock protein 70 (Hsp70) and the J-domain co-chaperone auxilin catalyse coat disassembly. However, a specific function of these factors in uncoating in vivo has not been demonstrated, leaving the physiological mechanism and significance of uncoating unclear. Here we report the identification and characterization of a Saccharomyces cerevisiae J-domain protein, Aux1. Inactivation of Aux1 results in accumulation of clathrin-coated vesicles, impaired cargo delivery, and an increased ratio of vesicle-associated to cytoplasmic clathrin. Our results demonstrate an in vivo uncoating function of a J domain co-chaperone and establish the physiological significance of uncoating in transport mediated by clathrin-coated vesicles.

Sanfilippo syndrome type B (mucopolysaccharidosis III B, MPS III B)is an autosomal recessive, neurodegenerative disease of children, characterized by profound mental retardation and dementia. The primary cause is mutation in the NAGLU gene, resulting in deficiency of ␣-N-acetylglucosaminidase and lysosomal accumulation of heparan sulfate. In the mouse model of MPS III B, neurons and microglia display the characteristic vacuolation of lysosomal storage of undegraded substrate, but neurons in the medial entorhinal cortex (MEC) display accumulation of several additional substances. We used whole genome microarray analysis to examine differential gene expression in MEC neurons isolated by laser capture microdissection from Naglu ؊/؊ and Naglu ؉/؊ mice. Neurons from the lateral entorhinal cortex (LEC) were used as tissue controls. The highest increase in gene expression (6-to 7-fold between mutant and control) in MEC and LEC neurons was that of Lyzs, which encodes lysozyme, but accumulation of lysozyme protein was seen in MEC neurons only. Because of a report that lysozyme induced the formation of hyperphosphorylated tau (Ptau) in cultured neurons, we searched for P-tau by immunohistochemistry. P-tau was found in MEC of Naglu ؊/؊ mice, in the same neurons as lysozyme. In older mutant mice, it was also seen in the dentate gyrus, an area important for memory. Electron microscopy of dentate gyrus neurons showed cytoplasmic inclusions of paired helical filaments, P-tau aggregates characteristic of tauopathies-a group of age-related dementias that include Alzheimer disease. Our findings indicate that the Sanfilippo syndrome type B should also be considered a tauopathy.hyperphosphorylated tau ͉ lysozyme ͉ mucopolysaccharidosis ͉ entorhinal cortex ͉ gene expression microarray

Highlights d Collagen V deficiency increases scar size after acute heart injury d Mechanical properties of scars are altered with Col V deficiency d Altered mechanosensitive cues augment myofibroblast formation in scar d Inhibition of specific integrins rescues increased scarring in Col-V-deficient states

The islet in type 2 diabetes (T2DM) and the brain in neurodegenerative diseases share progressive cell dysfunction, increased apoptosis, and accumulation of locally expressed amyloidogenic proteins (islet amyloid polypeptide (IAPP) in T2DM). Excessive activation of the Ca 2؉ -sensitive protease calpain-2 has been implicated as a mediator of oligomer-induced cell death and dysfunction in neurodegenerative diseases. To establish if human IAPP toxicity is mediated by a comparable mechanism, we overexpressed human IAPP in rat insulinoma cells and freshly isolated human islets. Pancreas was also obtained at autopsy from humans with T2DM and nondiabetic controls. We report that overexpression of human IAPP leads to the formation of toxic oligomers and increases beta cell apoptosis mediated by increased cytosolic Ca 2؉ and hyperactivation of calpain-2. Cleavage of ␣-spectrin, a marker of calpain hyperactivation, is increased in beta cells in T2DM. We conclude that overactivation of Ca 2؉ -calpain pathways contributes to beta cell dysfunction and apoptosis in T2DM. Hyperglycemia in type 2 diabetes mellitus (T2DM)3 is due to impaired insulin secretion in the setting of relative insulin resistance (1). The islets of Langerhans in T2DM are characterized by a deficit in beta cells, increased beta cell apoptosis, and islet amyloid derived from islet amyloid polypeptide (IAPP), a 37-amino acid highly conserved peptide co-expressed and secreted with insulin by pancreatic beta cells (2, 3).The pathology of the islet in T2DM and brain in neurodegenerative diseases such as Alzheimer disease share several parallels. In both, the loss of functional tissue is associated with deposition of a locally expressed protein with the potential to form amyloid fibrils (Alzheimer beta protein in Alzheimer disease and IAPP in T2DM) (2, 4). In both T2DM and Alzheimer disease, there has been a debate as to whether the amyloid deposits contribute to cell loss (the so-called amyloid hypothesis) or are secondary to the processes leading to cell loss. Evidence against a direct role of amyloid deposits on cell loss is the poor correlation between the extent of amyloid deposits and the severity of disease in both human and animal models (3,5,6). Moreover, preformed amyloid fibrils are not cytotoxic when applied to cells (7).However, several lines of evidence are supportive of a role of cytotoxicity by amyloidogenic proteins. These include genetic predisposition in occasional families with mutations leading to increased amyloidogenicity of the amyloid protein (8) and reproduction of the disease phenotype in rodent models transgenic for the relevant human amyloidogenic protein (9). There is an increasing appreciation that the cytotoxic forms of amyloidogenic proteins are small nonfibrillar oligomers that may form in membranes and cause nonselective membrane permeability (7, 10, 11), the toxic oligomer hypothesis. Moreover, misfolding and aggregation of amyloidogenic proteins into toxic oligomers induce apoptosis through the mechanism of endoplasmic retic...