MAB3418X Sigma-AldrichAnti-MAP2 Antibody, Alexa Fluor® 488 conjugated

One of the more profound features of systemic lupus erythematosus (SLE) is that females have a 9:1 prevalence of this disease over males. Up to 80% of SLE patients have cognitive defects or affective disorders. The mechanism of CNS injury responsible for cognitive impairment is unknown. We previously showed that ERα deficiency significantly reduced renal disease and increased survival in lupus-prone mice. We hypothesized that ERα deficiency would be similarly protective in the brain, and that ERα may play a role in modulating blood-brain barrier (BBB) integrity and/or neuroinflammation in lupus-prone mice.MRL/lpr ERα+/+ and ERαKO mice (n = 46) were ovariectomized, received 17β-estradiol pellets, and underwent radial arm water maze (WRAM) and novel object recognition (NOR) testing starting at eight weeks of age. Mice were sacrificed and brains were hemisected and processed for either immunohistochemistry, or hippocampus and parietal cortex dissection for Western blotting.MRL/lpr ERαKO mice (n = 21) performed significantly better in WRAM testing than wild-type MRL/lpr mice (n = 25). There was a significant reduction in reference memory errors (P less than 0.007), working memory errors (P less than 0.05), and start arm errors (P less than 0.02) in ERαKO mice. There were significant differences in NOR testing, particularly total exploration time, with ERα deficiency normalizing behavior. No significant differences were seen in markers of tight junction, astrogliosis, or microgliosis in the hippocampus or cortex by Western blot, however, there was a significant reduction in numbers of Iba1+ activated microglia in the hippocampus of ERαKO mice, as evidenced by immunohistochemietry (IHC).ERα deficiency provides significant protection against cognitive deficits in MRL/lpr mice as early as eight weeks of age. Additionally, the significant reduction in Iba1+ activated microglia in the MRL/lpr ERαKO mice was consistent with reduced inflammation, and may represent a biological mechanism for the cognitive improvement observed.

Infiltration of leukocytes into post-ischemic cerebrum is a well-described phenomenon in stroke injury. Because CD-8(+) T-lymphocytes secrete cytotoxic proteases, including granzyme-b (Gra-b) that exacerbates post-ischemic brain damage, we investigated roles of Gra-b in human stroke. To study the role of Gra-b in stroke, ischemic and non-ischemic tissues (from post-mortem stroke patients) were analyzed using immunoblotting, co-immunoprecipitation, terminal deoxy uridine nick end labeling (TUNEL) and Annexin-V immunostaining, and in vitro neuron survival assays. Activated CG-SH cells and supernatants were used to model leukocyte-dependent injury. Non-ischemic brain tissues were used as non-pathological controls. Non-activated CG-SH cells and supernatants were used as controls for in vitro experiments. Human stroke (ischemic) samples contained significantly higher levels of Gra-b and interferon-gamma inducible protein-10 (IP-10/CXCL10) than non-ischemic controls. In stroke, poly (ADP-ribose) polymerase-1 and heat shock protein-70 were cleaved to canonical proteolytic signature fragments by Gra-b. Gra-b was also found to bind to Bid and caspase-3. Gra-b also co-localized with Annexin-V(+) /TUNEL(+) in degenerating neurons. Importantly, Gra-b inhibition protected both normal and ischemia-reperfused neurons against in vitro neurotoxicity mediated by activated CG-SH cells and supernatants. These results suggest that increased leukocyte infiltration and elevated Gra-b levels in the post-stroke brain can induce contact-dependent and independent post-ischemic neuronal death to aggravate stroke injury.

The physiological responses of AMPA receptors can be modulated through the differential expression of their subunits and by modifying their number at the cell surface. Here we have studied the expression of AMPA receptor subunits (GluR1-4) mRNAs in cerebellar granule cells grown in depolarizing (25mMK(+)) medium, and we have evaluated the effect of decreasing the [K(+)] in the culture medium for 24 h on both GluR1-4 expression (both mRNA and protein) and their presence at the plasma membrane. The expression of the four AMPAR subunits increases as the [K(+)] decreases, although the increase in GluR2 and GluR3 was only observed in the cell soma but not in the dendrites. Calcium entry through L-type calcium channel and CaMKIV activation are responsible for the reduction in the expression of AMPA receptor subunits in cells cultured in depolarizing conditions. Indeed, prolonged reduction of extracellular [K(+)] or blockage of L-type calcium channels enhanced both the surface insertion of the four AMPAR subunits and the AMPA response measured through intracellular calcium increase. These findings reveal a balanced increase in functional AMPA receptors at the surface of cells that can trigger strong increases in calcium in response to the persistent reduction of calcium entry.

Using a monoclonal antibody against the microtubule-associated protein tau we compared the distribution and the biochemical maturation of this protein in hippocampal pyramidal neurons in the rat in tau and in culture. In tissue sections from mature animals tau was localized heterogeneously within neurons. It was concentrated in axons; dendrites and somata showed little or no staining. In hippocampal cultures ranging from 12 h to 4 weeks in vitro tau was present in neurons but not in glial cells, as it is in situ. Within cultured neurons, however, tau was not compartmentalized but was present throughout the dendrites, axons and somata. Immunoblotting experiments showed that the biochemical maturation of tau that occurs in situ also failed to occur in culture. The young form of tau persisted, and the adult forms did not develop. In contrast the biochemical maturation and the compartmentalization of microtubule-associated protein 2 occurred normally in hippocampal cultures. These results show that the biochemical maturation and the intraneuronal compartmentalization of these two microtubule-associated proteins are independently controlled. Despite the non-restricted distribution of tau in hippocampal neurons in culture, and despite the presence of only the immature isoform which has a lessened stimulatory effect on microtubule polymerization, axons and dendrites appear to grow normally and to exhibit appropriate functional properties.

In dissociated-cell cultures prepared from the embryonic rat hippocampus, neurons establish both axons and dendrites, which differ in geometry, in ultrastructure, and in synaptic polarity. We have used immunocytochemistry with monoclonal antibodies to study the regional distribution of beta-tubulin and micro-tubule-associated protein 2 (MAP2) in hippocampal cultures and their localization during early stages of axonal and dendritic development. After development for a week or more in culture, when axons and dendrites were well-differentiated, the distribution of these two proteins was quite different. Beta-tubulin was present throughout the nerve cell, in soma, dendrites, and axon. It was also present in all classes of non-neuronal cells, astrocytes, fibroblasts, and a presumptive glial progenitor cell. In contrast, MAP2 was preferentially localized to nerve cells; within neurons, MAP2 was present in soma and dendrites, but little or no immunostaining was detectable in axons. Both beta-tubulin and MAP2 were present in nerve cells at the time of plating. From the earliest stages of process extension, beta-tubulin was present in all neuronal processes, both axons and dendrites. Surprisingly, MAP2 was also initially present in both axons and dendrites, extending as far as the axonal growth cone. With subsequent development, MAP2 staining was selectively lost from the axon so that after 1 week in vitro little or no axonal staining remained. Taken together with earlier results (Cáceres et al., 1984a), these data indicate that the establishment of neuronal polarity, as manifested by the molecular differentiation of the axonal and dendritic cytoskeleton, occurs largely under endogenous control, even under culture conditions in which cell interactions are greatly restricted.(ABSTRACT TRUNCATED AT 250 WORDS)

The electrophoretic pattern of the large microtubule-associated protein, MAP2, changes during rat brain development. Immunoblots of NaDodSO4 extracts obtained from the cerebral cortex, cerebellum, and thalamus at 10-15 days after birth reveal only a single electrophoretic species when probed with any of three MAP2 monoclonal antibodies. By contrast, adult MAP2 contains two immunoreactive species, MAP2a and MAP2b. The single band of MAP2 from immature brain electrophoretically comigrates with adult MAP2b. Between postnatal days 17 and 18, immature MAP2 simultaneously resolves into two species in both the cerebellum and cerebral cortex. Immunoblots of NaDodSO4 extracts from spinal cord demonstrate the adult complement of MAP2 by day 10, indicating that MAP2 does not change coordinately throughout the entire central nervous system. In vitro cAMP-dependent phosphorylation of immature MAP2 causes a band split reminiscent of that seen during brain development in vivo. The possibility that the developmentally regulated changes observed in MAP2 during brain maturation are due to timed phosphorylation events is discussed.

The distribution and subcellular localization of tubulin and MAP2 in brain tissue were analyzed by immunocytochemistry with monoclonal hybridoma antibodies prepared against Chinese hamster brain tubulin and MAP2. We examined three anti-tubulin hybridoma antibodies (Tu3B, Tu9B, Tu12) specific for beta-tubulin, and two anti-MAP2 hybridoma antibodies (AP9,AP13). The specificity of each of the monoclonal antibodies was characterized by staining nitrocellulose electrophoretic blots of SDS-polyacrylamide gels of whole brain or hippocampal extracts. Each hybridoma antibody bound only its respective antigen in these preparations. Polyclonal antisera against tubulin were also examined. Sections reacted with antisera against tubulin or monoclonal antibodies against beta-tubulin revealed a wide variety of stained cellular compartments. The reaction product was found to decorate dendritic and axonal microtubles in neurons; glial cells were also stained. MAP2 immunoreactivity was found only in neurons. In the case of one of the monoclonal antibodies (AP9), staining was preferentially associated with dendritic processes. However, light but significant staining of axonal processes was seen with AP13. Within dendrites, MAP2 was found associated with dendritic microtubules and postsynaptic densities (psd), both in shaft and spine synapses. In addition, strong immunoreactivity for MAP2 was found within the cytoplasm of dendritic spines. There was little or no immunoreactivity for tubulin in the spine cytoplasm, although the psd was stained. The localization of MAP2 in dendritic spines and in the psd suggests that this protein may have a biological role independent of its association with microtubules. The observations on differential staining of the hybridoma antibodies against MAP2 suggest that there may be distinct subtypes or states of MAP2 within neurons.

A method has been devised for the electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. The method results in quantitative transfer of ribosomal proteins from gels containing urea. For sodium dodecyl sulfate gels, the original band pattern was obtained with no loss of resolution, but the transfer was not quantitative. The method allows detection of proteins by autoradiography and is simpler than conventional procedures. The immobilized proteins were detectable by immunological procedures. All additional binding capacity on the nitrocellulose was blocked with excess protein; then a specific antibody was bound and, finally, a second antibody directed against the first antibody. The second antibody was either radioactively labeled or conjugated to fluorescein or to peroxidase. The specific protein was then detected by either autoradiography, under UV light, or by the peroxidase reaction product, respectively. In the latter case, as little as 100 pg of protein was clearly detectable. It is anticipated that the procedure will be applicable to analysis of a wide variety of proteins with specific reactions or ligands.