Validation of Flow Cytometry and Magnetic Bead-Based Methods to Enrich CNS Single Cell Suspensions for Quiescent Microglia.

Microglia are resident mononuclear phagocytes within the CNS parenchyma that intimately interact with neurons and astrocytes to remodel synapses and extracellular matrix. We briefly review studies elucidating the molecular pathways that underlie microglial surveillance, activation, chemotaxis, and phagocytosis; we additionally place these studies in a clinical context. We describe and validate an inexpensive and simple approach to obtain enriched single cell suspensions of quiescent parenchymal and perivascular microglia from the mouse cerebellum and hypothalamus. Following preparation of regional CNS single cell suspensions, we remove myelin debris, and then perform two serial enrichment steps for cells expressing surface CD11b. Myelin depletion and CD11b enrichment are both accomplished using antigen-specific magnetic beads in an automated cell separation system. Flow cytometry of the resultant suspensions shows a significant enrichment for CD11b(+)/CD45(+) cells (perivascular microglia) and CD11b(+)/CD45(-) cells (parenchymal microglia) compared to starting suspensions. Of note, cells from these enriched suspensions minimally express Aif1 (aka Iba1), suggesting that the enrichment process does not evoke significant microglial activation. However, these cells readily respond to a functional challenge (LPS) with significant changes in the expression of molecules specifically associated with microglia. We conclude that methods employing a combination of magnetic-bead based sorting and flow cytometry produce suspensions highly enriched for microglia that are appropriate for a variety of molecular and cellular assays.

Intramembrane charge movements and excitation- contraction coupling expressed by two-domain fragments of the Ca2+ channel.

To investigate the molecular basis of the voltage sensor that triggers excitation-contraction (EC) coupling, the four-domain pore subunit of the dihydropyridine receptor (DHPR) was cut in the cytoplasmic linker between domains II and III. cDNAs for the I-II domain (alpha1S 1-670) and the III-IV domain (alpha1S 701-1873) were expressed in dysgenic alpha1S-null myotubes. Coexpression of the two fragments resulted in complete recovery of DHPR intramembrane charge movement and voltage-evoked Ca(2+) transients. When fragments were expressed separately, EC coupling was not recovered. However, charge movement was detected in the I-II domain expressed alone. Compared with I-II and III-IV together, the charge movement in the I-II domain accounted for about half of the total charge (Q(max) = 3 +/- 0.23 vs. 5.4 +/- 0.76 fC/pF, respectively), and the half-activation potential for charge movement was significantly more negative (V(1/2) = 0.2 +/- 3.5 vs. 22 +/- 3.4 mV, respectively). Thus, interactions between the four internal domains of the pore subunit in the assembled DHPR profoundly affect the voltage dependence of intramembrane charge movement. We also tested a two-domain I-II construct of the neuronal alpha1A Ca(2+) channel. The neuronal I-II domain recovered charge movements like those of the skeletal I-II domain but could not assist the skeletal III-IV domain in the recovery of EC coupling. The results demonstrate that a functional voltage sensor capable of triggering EC coupling in skeletal myotubes can be recovered by the expression of complementary fragments of the DHPR pore subunit. Furthermore, the intrinsic voltage-sensing properties of the alpha1A I-II domain suggest that this hemi-Ca(2+) channel could be relevant to neuronal function.

The mouse stargazer gene encodes a neuronal Ca2+-channel gamma subunit.

Stargazer mice have spike-wave seizures characteristic of absence epilepsy, with accompanying defects in the cerebellum and inner ear. We describe here a novel gene, Cacng2, whose expression is disrupted in two stargazer alleles. It encodes a 36-kD protein (stargazin) with structural similarity to the gamma subunit of skeletal muscle voltage-gated calcium (Ca2+) channels. Stargazin is brain-specific and, like other neuronal Ca2+-channel subunits, is enriched in synaptic plasma membranes. In vitro, stargazin increases steady-state inactivation of alpha1 class A Ca2+ channels. The anticipated effect in stargazer mutants, inappropriate Ca2+ entry, may contribute to their more pronounced seizure phenotype compared with other mouse absence models with Ca2+-channel defects. The discovery that the stargazer gene encodes a gamma subunit completes the identification of the major subunit types for neuronal Ca2+ channels, namely alpha1, alpha2delta, beta and gamma, providing a new opportunity to understand how these channels function in the mammalian brain and how they may be targeted in the treatment of neuroexcitability disorders.

Evidence for a 95 kDa short form of the alpha1A subunit associated with the omega-conotoxin MVIIC receptor of the P/Q-type Ca2+ channels.

Neuronal voltage-dependent Ca2+ channels have been isolated previously and shown to contain a primary alpha1 pore-forming subunit as well as auxiliary alpha2delta and beta subunits, in addition to an uncharacterized 95 kDa protein. In the present study, using multiple approaches, we have extensively characterized the molecular structure of the 95 kDa protein. Separation of the P/Q- and N-type neuronal Ca2+ channels showed that the 95 kDa protein is associated exclusively with the omega-Conotoxin MVIIC receptor of the P/Q-type channels. Analysis of purified synaptic plasma membranes and the isolated P/Q-type channels, using alpha1A-specific antibodies, suggested a structural relationship between the alpha1A subunit and the 95 kDa protein. This finding was supported by protein-protein interaction data, which revealed that the beta subunit can associate with the 95 kDa protein in addition to the alpha1A subunit. Changes in electrophoretic mobility after enzymatic treatment with Endo F indicated that the 95 kDa protein is glycosylated. Furthermore, microsequencing of the 95 kDa protein yielded 13 peptide sequences, all of which are present in the first half of the alpha1A subunit up to amino acid 829 of the cytoplasmic linker between repeats II and III. Taken together, our results strongly suggest that the 95 kDa glycoprotein associated with the P/Q-type Ca2+ channels is a short form of the alpha1A subunit.