Targeted deletion of βIII spectrin impairs synaptogenesis and generates ataxic and seizure phenotypes

Abstract

The spectrin membrane skeleton controls the disposition of selected membrane channels, receptors, and transporters. In the brain βIII spectrin binds directly to the excitatory amino acid transporter (EAAT4), the glutamate receptor delta, and other proteins. Mutations in βIII spectrin link strongly to human spinocerebellar ataxia type 5 (SCA5), correlating with alterations in EAAT4. We have explored the mechanistic basis of this phenotype by targeted gene disruption of Spnb3. Mice lacking intact βIII spectrin develop normally. By 6 months they display a mild nonprogressive ataxia. By 1 year most Spnb3 −/− animals develop a myoclonic seizure disorder with significant reductions of EAAT4, EAAT1, GluRδ, IP3R, and NCAM140. Other synaptic proteins are normal. The cerebellum displays increased dark Purkinje cells (PC), a thin molecular layer, fewer synapses, a loss of dendritic spines, and a 2-fold expansion of the PC dendrite diameter. Membrane and expanded Golgi profiles fill the PC dendrite and soma, and both regions accumulate EAAT4. Correlating with the seizure disorder are enhanced hippocampal levels of neuropeptide Y and EAAT3 and increased calpain proteolysis of αII spectrin. It appears that βIII spectrin disruption impairs synaptogenesis by disturbing the intracellular pathways selectively regulating protein trafficking to the synapse. The mislocalization of these proteins secondarily disrupts glutamate transport dynamics, leading to seizures, neuronal damage, and compensatory changes in EAAT3 and neuropeptide Y.cytoskeleton | membrane | spinocerebellar ataxia type 5 | excitatory amino acid transporter 4 | Purkinje T he mammalian nervous system expresses seven spectrin genes, two encoding α-subunits and five encoding β-subunits. All are large multifunctional molecules, and all display distinctive cellular and subcellular distributions. Their role in neuronal cells remains largely conjectural. Although generally thought to organize the membrane surface or to bestow membrane stability, their diversity implies a function beyond simple stabilization. Studies in cultured cells reveal spectrin and ankyrin as scaffolds organizing internal organelles (1-6) or receptor clusters (7-10). These proteins also facilitate protein transport in the secretory (3, 10, 11) and endocytic pathways (12-14) (reviewed in ref. 15). Consonant with these roles are observations that genetic deletion or mutation of spectrins or ankyrins may cause missorting of unique subsets of adhesion molecules, receptors, and ion channels in brain or muscle, with consequential cardiovascular, neuromuscular, and neurodegenerative disease (16-18).In the nervous system, cortical (19) and cerebellar granular cell neurons (7) are enriched in βI spectrin, especially at the postsynaptic density (PSD) and on a subset of vesicular organelles. Spectrin βII is associated with axonal processes (20), and βIV spectrin is concentrated at the nodes of Ranvier and along the initial axon segment (21). Less is known about βIII spectrin, although studies point to an intere...

Structural organization of the actin-spectrin–based membrane skeleton in dendrites and soma of neurons

“…Because βIII-spectrin is the isoform of β-spectrin that is specifically enriched in dendrites (26,27), it is possible that βIII-spectrin is the major β-spectrin component of the dendritic MPS structure, and that βII-spectrin is not essential for the formation of this structure in dendrites. To test this possibility, we knocked down βII-spectrin in DIV 28 mouse neurons, using a shRNA-expressing adenovirus (28).…”

“…We used 3D STORM imaging (23,24) to examine the distributions of βII-spectrin, βIII-spectrin, actin, and adducin in dendrites and axons of cultured mouse hippocampal neurons. Actin, adducin and βII-spectrin are abundantly expressed in both axons and dendrites (9,25), whereas βIII-spectrin is a β-spectrin isoform specifically enriched in dendrites with only a low expression level in axons (26,27). We used immunofluorescence to label βII-spectrin, βIII-spectrin, and adducin, and dyeconjugated phalloidin to label actin.…”

Cell organization, growth, and neural and cardiac development require αII-spectrin

“…In lymphocytes, the trafficking of receptors CD3 and CD45 are both spectrin-and ankyrin-dependent (Pradhan and Morrow, 2002). Mutations in bIII-spectrin cause spinocerebellar ataxia type 5 in humans (Ikeda et al, 2006) (and ataxia and seizures in mice) Stankewich et al, 2010) due to impaired intracellular transport of EAAT4 and at least five other proteins associated with the synapse Stankewich et al, 2010). Thus, given that the Spna2 2/2 mice are deficient in and mis-localize their ankyrins and bII-and bIII-spectrin, it is likely that in these animals there are also problems with membrane protein organization and sorting.…”

Annexin B9 binds to βH-spectrin and is required for multivesicular body function in Drosophila

“…Roles for the SBMS have been demonstrated in multiple organisms and tissues including: in neuronal structure, function and membrane organization (Hammarlund et al, 2007;Hulsmeier et al, 2007;Ikeda et al, 2006;Lacas-Gervais et al, 2004;Pielage et al, 2006); in epithelial structure and stability (Lee et al, 2010;Lee et al, 1997;Thomas et al, 1998); and in muscle function (Bennett and Healy, 2008;Mohler et al, 2005). Widely regarded as a static structural element, emerging results have indicated that these proteins actually have dynamic roles in transport processes including: trans-Golgi network to plasma membrane (Kizhatil et al, 2007a); ER to Golgi (Stabach et al, 2008;Stankewich et al, 2010); at the early endosome (Phillips and Thomas, 2006); and endosome to lysosome transport (Johansson et al, 2007). Some of these roles arise through its interaction with the dynactin complex (Johansson et al, 2007;Lorenzo et al, 2010;Muresan et al, 2001), and together these data suggest that regulation of trafficking is another core function of the spectrins.…”