MAB362 Sigma-AldrichAnti-MAP1 Antibody, clone HM-1

2,5-Hexanedione (HD) intoxication is associated with axon atrophy that might be responsible for the characteristic gait abnormalities, hindlimb skeletal muscle weakness and other neurological deficits that accompany neurotoxicity. Although previous mechanistic research focused on neurofilament triplet proteins (NFL, NFM, NFH), other cytoskeletal targets are possible. Therefore, to identify potential non-NF protein targets, we characterized the effects of HD on protein-protein interactions in cosedimentation assays using microtubules and NFs prepared from spinal cord of rats intoxicated at different daily dose rates (175 and 400 mg/kg/day). Results indicate that HD did not alter the presence of alpha- or beta-tubulins in these preparations, nor were changes noted in the distribution of either anterograde (KIF1A, KIF3, KIF5) or retrograde (dynein) molecular motors. The cosedimentation of dynactin, a dynein-associated protein, also was not affected. Immunoblot analysis of microtubule-associated proteins (MAPs) in microtubule preparations revealed substantial reductions (45-80%) in MAP1A, MAP1B heavy chain, MAP2, and tau regardless of HD dose rate. MAP1B light chain content was not altered. Finally, HD intoxication did not influence native NF protein content in either preparation. As per previous research, microtubule and NF preparations were enriched in high-molecular weight NF species. However, these NF derivatives were common to both HD and control samples, suggesting a lack of pathognomonic relevance. These data indicate that, although motor proteins were not affected, HD selectively impaired MAP-microtubule binding, presumably through adduction of lysine residues that mediate such interactions. Given their critical role in cytoskeletal physiology, MAPs could represent a relevant target for the induction of gamma-diketone axonopathy.

PURPOSE: Cytoskeleton proteins play a critical role in maintaining retinal ganglion cell structure, viability, and function. This study documents the distribution of cytoskeleton protein subunits in the various regions of the normal human optic nerve and identifies important relationships among mitochondria, myelin, and neurofilament proteins. METHODS: Twenty-three optic nerves from human cadavers were used. Confocal microscopy was used to examine the distribution of neurofilament light, neurofilament medium, neurofilament heavy (phosphorylated and unphosphorylated), neurofilament heavy (phosphorylated only), actin, and microtubule associated protein (MAP)-1 along the sagittal plane of the optic nerve. Comparisons were made among superior, middle, and inferior regions and also among temporal, central, and nasal portions of the optic nerve. Colocalization of neurofilament light, mitochondrial cytochrome c oxidase (COX), and myelin was also performed. RESULTS: There are significant differences in the pattern and distribution of neurofilament protein subunits, actin, and MAP-1 along the sagittal plane of the optic nerve. Cytoskeleton proteins and COX mitochondria are found in highest concentrations in the prelaminar and lamina cribrosa regions. COX and neurofilament light occurs predominantly in unmyelinated nerve, with a significant decrease in concentration occurring on optic nerve myelination. CONCLUSIONS: The heterogeneous distribution of cytoskeleton proteins along the sagittal plane may be an important functional adaptation that reflects the nonuniform nature of the physiological and structural environment of the optic nerve. The heterogeneous distribution of cytoskeleton proteins may also partly account for the asymmetric pattern of optic nerve damage after intraocular pressure elevation.

Large-conductance voltage- and calcium-sensitive channels are known to be expressed in the plasmalemma of central neurons; however, recent data suggest that large-conductance voltage- and calcium-sensitive channels may also be present in mitochondrial membranes. To determine the subcellular localization and distribution of large-conductance voltage- and calcium-sensitive channels, rat brain fractions obtained by Ficoll-sucrose density gradient centrifugation were examined by Western blotting, immunocytochemistry and immuno-gold electron microscopy. Immunoblotting studies demonstrated the presence of a consistent signal for the alpha subunit of the large-conductance voltage- and calcium-sensitive channel in the mitochondrial fraction. Double-labeling immunofluorescence also demonstrated that large-conductance voltage- and calcium-sensitive channels are present in mitochondria and co-localize with mitochondrial-specific proteins such as the translocase of the inner membrane 23, adenine nucleotide translocator, cytochrome c oxidase or complex IV-subunit 1 and the inner mitochondrial membrane protein but do not co-localize with calnexin, an endoplasmic reticulum marker. Western blotting of discrete subcellular fractions demonstrated that cytochrome c oxidase or complex IV-subunit 1 was only expressed in the mitochondrial fraction whereas actin, acetylcholinesterase, cadherins, calnexin, 58 kDa Golgi protein, lactate dehydrogenase and microtubule-associated protein 1 were not, demonstrating the purity of the mitochondrial fraction. Electron microscopic examination of the mitochondrial pellet demonstrated gold particle labeling within mitochondria, indicative of the presence of large-conductance voltage- and calcium-sensitive channels in the inner mitochondrial membrane. These studies provide concrete morphological evidence for the existence of large-conductance voltage- and calcium-sensitive channels in mitochondria: our findings corroborate the recent electrophysiological evidence of mitochondrial large-conductance voltage- and calcium-sensitive channels in glioma and cardiac cells.

The distribution of microtubule-associated proteins (MAPs) 1 and 2 in rat brain was studied using monoclonal antibodies. Immunochemical staining showed that both MAP1 and MAP2 are present only in neurons and both are highly concentrated in dendrites compared to axons. Otherwise, they differed in distribution in various ways. MAP1 was present at low levels in axons, whereas MAP2 was never detectable in axons with either of two different fixation methods used. In the cerebellum the two MAPs differed in relative concentration in various classes on neurons. Thus, anti-MAP1 staining was strong in Purkinje cells but very faint in granule cells, whereas anti-MAP2 staining was strong in both. There were also distributional differences within the same cell. Thus, in Purkinje cells, anti-MAP1 staining is strong in the cell body, initial axon segment and throughout the dendritic tree, but anti-MAP2 staining is present only in dendrites beyond the initial proximal portion. These results suggest that microtubules with different molecular compositions are present in the cerebellum where they are distributed differently between cells as well as within the same cell.