Clearance of extracellular and cell-associated amyloid beta peptide through viral expression of neprilysin in primary neurons.

Amyloid beta peptide (Abeta), the pathogenic agent of Alzheimer's disease (AD), is a physiological metabolite constantly anabolized and catabolized in the brain. We previously demonstrated that neprilysin is the major Abeta-degrading enzyme in vivo. To investigate whether or not manipulation of neprilysin activity in the brain would be an effective strategy for regulating Abeta levels, we expressed neprilysin in primary cortical neurons using a Sindbis viral vector and examined the effect on Abeta metabolism. The corresponding recombinant protein, expressed in the cell bodies and processes, exhibited thiorphan-sensitive endopeptidase activity, whereas a mutant neprilysin with an amino acid substitution in the active site did not show any such activity. Expression of the wild-type neprilysin, but not the mutant, led to significant decreases in both the Abeta40 and 42 levels in the culture media in a dose-dependent manner. Moreover, neprilysin expression also resulted in reducing cell-associated Abeta, which could be more neurotoxic than extracellular Abeta. These results indicate that the manipulation of neprilysin activity in neurons, the major source of Abeta in the brain, would be a relevant strategy for controlling the Abeta levels and thus the Abeta-associated pathology in brain tissues.

Matrix metalloproteinase (MMP) system in brain: identification and characterization of brain-specific MMP highly expressed in cerebellum.

The matrix metalloproteinase (MMP) family, comprising more than 20 isoforms, modulates the extracellular milieu by degrading extracellular matrix (ECM) proteins. Because MMP is one of the few groups of proteinases capable of hydrolysing insoluble fibrillar proteins, they are likely to play crucial roles in regulating both normal and pathophysiological processes in the brain. However, little is yet known about their possible neuronal functions due presumably to their unusual redundancy and to the absence of a complete catalogue of isoforms. As an initial step in understanding the MMP system in the brain, we analysed an expression spectrum of MMP in rat brain using RT-PCR and discovered a novel brain-specific MMP, MT5-MMP. MT5-MMP was the predominant species among the nongelatinase-type isoforms in brain. MT5-MMP, present in all brain tissues examined, was most strongly expressed in cerebellum and was localized in the membranous structures of expressing neurons, as assessed biochemically and immunohistochemically. In cerebellum, its expression was regulated developmentally and was closely associated with dendritic tree formation of Purkinje cells, suggesting that MT5-MMP may contribute to neuronal development. Furthermore, its stable postdevelopmental expression and colocalization with senile plaques in Alzheimer brain indicates possible roles in neuronal remodeling naturally occurring in adulthood and in regulating pathophysiological processes associated with advanced age.

Phosphorylation of cofilin by LIM-kinase is necessary for semaphorin 3A-induced growth cone collapse.

Semaphorin 3A is a chemorepulsive axonal guidance molecule that depolymerizes the actin cytoskeleton and collapses growth cones of dorsal root ganglia neurons. Here we investigate the role of LIM-kinase 1, which phosphorylates an actin-depolymerizing protein, cofilin, in semaphorin 3A-induced growth cone collapse. Semaphorin 3A induced phosphorylation and dephosphorylation of cofilin at growth cones sequentially. A synthetic cell-permeable peptide containing a cofilin phosphorylation site inhibited LIM-kinase in vitro and in vivo, and essentially suppressed semaphorin 3A-induced growth cone collapse. A dominant-negative LIM kinase, which could not be activated by PAK or ROCK, suppressed the collapsing activity of semaphorin 3A. Phosphorylation of cofilin by LIM-kinase may be a critical signaling event in growth cone collapse by semaphorin 3A.

Identification of the major Abeta1-42-degrading catabolic pathway in brain parenchyma: suppression leads to biochemical and pathological deposition.

Alzheimer amyloid beta-peptide (Abeta) is a physiological peptide constantly anabolized and catabolized under normal conditions. We investigated the mechanism of catabolism by tracing multiple-radiolabeled synthetic peptide injected into rat hippocampus. The Abeta1-42 peptide underwent full degradation through limited proteolysis conducted by neutral endopeptidase (NEP) similar or identical to neprilysin as biochemically analyzed. Consistently, NEP inhibitor infusion resulted in both biochemical and pathological deposition of endogenous Abeta42 in brain. This NEP-catalyzed proteolysis therefore limits the rate of Abeta42 catabolism, up-regulation of which could reduce the risk of developing Alzheimer's disease by preventing Abeta accumulation.

MuSC, a novel member of the immunoglobulin superfamily, is expressed in neurons of a subset of cranial sensory ganglia in the mouse embryo.

In contrast to the spinal sensory ganglia which reiterate a basic organizational and functional unit, each cranial ganglion mediates a distinct sensory modality and exhibits a characteristic pattern of peripheral and central neuronal connectivity. Molecules responsible for establishment and maintenance of the cranial ganglion-specific networks are not known. Our hamster monoclonal antibody 802C11 strongly stained neurons and their processes of the VIIIth cranial ganglion (hearing and equilibrium), but not of the Vth cranial (somatosensory) or spinal ganglia in the mouse embryo. The cellular staining pattern of positive neurons suggested that the antigen was associated with the cell membrane, and biochemical analyses of the antigen from adult mouse brain showed the antigen to be a glycosylated intrinsic membrane protein of approximately 100 kDa. The antigen was purified, and based on the partial amino acid sequences, its entire cDNA was cloned. A bacterially expressed polypeptide encoded by the cDNA was recognized by the antibody. The deduced amino acid sequence revealed that the antigen belongs to the immunoglobulin superfamily with a significant homology (73.5% identity) to chicken SC1 protein. Chicken SC1 has been shown to be a cell-cell adhesion molecule in vitro with a proposed role in neurite extension of spinal motor neurons. These results suggest that our murine SC1-related protein (MuSC) is involved in the pathfinding and/or fasciculation of specific cranial sensory nerve fibres.

The neuron-specific kinesin superfamily protein KIF1A is a unique monomeric motor for anterograde axonal transport of synaptic vesicle precursors.

Axonal transport has been intensively examined as a good model for studying the mechanism of organelle transport in cells, but it is still unclear how different types of membrane organelles are transported through the nerve axon. To elucidate the function of this mechanism, we have cloned KIF1A, a novel neuron-specific kinesin superfamily motor that was discovered to be a monomeric, globular molecule and that had the fastest reported anterograde motor activity (1.2 microns/s). To identify its cargo, membranous organelles were isolated from the axon. KIF1A was associated with organelles that contained synaptic vesicle proteins such as synaptotagmin, synaptophysin, and Rab3A. However, this organelle did not contain SV2, another synaptic vesicle protein, nor did it contain presynaptic membrane proteins, such as syntaxin 1A or SNAP-25, or other known anterograde motor proteins, such as kinesin and KIF3. Thus, we suggest that the membrane proteins are sorted into different classes of transport organelles in the cell body and are transported by their specific motor proteins through the axon.