Faculty Bibliography

The identification of molecular motors that modulate the neuronal cytoskeleton has been elusive. Here, we show that a molecular motor protein, myosin Va, is present in high proportions in the cytoskeleton of mouse CNS and peripheral nerves. Immunoelectron microscopy, coimmunoprecipitation, and blot overlay analyses demonstrate that myosin Va in axons associates with neurofilaments, and that the NF-L subunit is its major ligand. A physiological association is indicated by observations that the level of myosin Va is reduced in axons of NF-L-null mice lacking neurofilaments and increased in mice overexpressing NF-L, but unchanged in NF-H-null mice. In vivo pulse-labeled myosin Va advances along axons at slow transport rates overlapping with those of neurofilament proteins and actin, both of which coimmunoprecipitate with myosin Va. Eliminating neurofilaments from mice selectively accelerates myosin Va translocation and redistributes myosin Va to the actin-rich subaxolemma and membranous organelles. Finally, peripheral axons of dilute-lethal mice, lacking functional myosin Va, display selectively increased neurofilament number and levels of neurofilament proteins without altering axon caliber. These results identify myosin Va as a neurofilament-associated protein, and show that this association is essential to establish the normal distribution, axonal transport, and content of myosin Va, and the proper numbers of neurofilaments in axons

The calcium-activated protease calpain cleaves a variety of biologically important proteins and serves, therefore, as a key regulator of many cellular functions. Activation of both main isoforms, calpain 1 and calpain 2, was demonstrated previously in Alzheimer's disease. In this report, antibodies specifically recognizing the active form of calpain 2 were used to investigate calpain 2 activation in a broad range of neurodegenerative diseases, utilizing multiple-label confocal immunofluorescence imaging. With rare exceptions, the active form of calpain 2 was found in colocalization with hyperphosphorylated tau protein. Aggregates of mutated huntingtin, alpha-synuclein, or unidentified protein in motor neuron disease type of frontotemporal dementia were always negative. These findings indicate that calpain 2 activation is not a general response to protein aggregation. In tauopathies, more pathological inclusions were labeled for hyperphosphorylated tau than for activated calpain 2. The extent of colocalization varied in both a disease-specific and cell-type specific manner. The active form of calpain 2 was detected in 50-75% of tau neurofibrillary pathology in Alzheimer's disease, Alzheimer neurofibrillary changes and Down's syndrome, as well as in the accompanying Alzheimer-type tau pathology in diffuse Lewy bodies disease, progressive supranuclear palsy, and corticobasal degeneration. For glial cells, only 10-25% of tuft-shaped astrocytes, glial plaques, or coiled bodies contained activated calpain 2. The majority of Pick bodies were negative. The association of calpain 2 activation with hyperphosphorylated tau might be the result of an attempt by the calpain proteolytic system to degrade the tau protein aggregates. Alternatively, calpain 2 could be directly involved in tau hyperphosphorylation by modulating protein kinase activities. Overall, these results provide evidence of the important role of the calpain proteolytic system in the pathogenesis of neurodegenerative diseases with tau neurofibrillary pathology

The lysosomal system (LS) is markedly activated in vulnerable neuronal populations early in Alzheimer's disease, although lysosomes become less efficient degradative compartments as neurons become more compromised. LS dysfunction, especially altered activity of the lysosomal protease cathepsin D, has been implicated in cell death initiation under various apoptotic conditions in vivo and in vitro. In this study, we observed that cathepsin D content increases nearly 3-fold in the human neocortex during normal aging while lysosomal cysteine protease activities decrease. By contrast, during aging in the mouse, this protease imbalance and other aging-related changes of the LS, such as lipofuscin accumulation, are minimal in these cortical areas. However, when leupeptin (a cysteine protease inhibitor) was infused intraventricularly, an imbalance of cathepsins similar to that in the aging human brain was induced. This was accompanied by changes associated with cell senescence, including ceroid-lipofuscin accumulation and alterations of tau proteolysis. In PSM146L/APPSWE transgenic mice, super-imposition of this aging-related cathepsin imbalance accentuated preexisting LS abnormalities to the level seen in AD brain and also induced neuronal atrophy and neurodegeneration. The minimal degree of 'lysosomal aging' seen in old mice, compared to that in humans, may partly explain the mild neurodegenerative phenotypes in transgenic models of AD pathology. In addition, these studies provide in vivo evidence relating altered lysosomal function to neurodegeneration