Sequential processing of amyloid precursor protein (APP) by membrane-bound proteases, BACE1 and ␥-secretase, plays a crucial role in the pathogenesis of Alzheimer disease. Much has been discovered on the properties of these proteases; however, regulatory mechanisms of enzyme-substrate interaction in neurons and their involvement in pathological changes are still not fully understood. It is mainly because of the membrane-associated cleavage of these proteases and the lack of information on new substrates processed in a similar way to APP. Here, using RNA interference-mediated BACE1 knockdown, mouse embryonic fibroblasts that are deficient in either BACE1 or presenilins, and BACE1-deficient mouse brain, we show clear evidence that ␤ subunits of voltage-gated sodium channels are sequentially processed by BACE1 and ␥-secretase. These results may provide new insights into the underlying pathology of Alzheimer disease.Alzheimer disease is a progressive neurodegenerative disorder and the most common form of age-dependent dementia. The major pathological features of Alzheimer disease are senile plaques and neurofibrillary tangles, which are the deposits of amyloid ␤ peptide (A␤) 1 and hyperphosphorylated tau, respectively. It is widely accepted that the sequential processing of APP, a type I membrane protein, by ␤-and ␥-secretases in the brain is crucial for the accumulation of A␤ and disease pathogenesis (1, 2). Although ␤-site APP-cleaving enzyme (BACE1) has been identified to be the ␤-secretase (3-6), a growing body of evidence favors presenilins-1 and -2 as the catalytic core of ␥-secretase (7). Although the properties of both proteases as APP processing enzymes are relatively well established, the regulatory mechanisms of sequential cleavage by both proteases in neurons are not completely clear. This is partly because of the fact that APP and its family proteins are still the only substrates identified for both ␤-and ␥-secretases, although a number of integral membrane proteins have been reported to be processed either by BACE1 (8, 9) or ␥-secretase (10). Identifying new substrates for both ␤-and ␥-secretases in neurons would therefore be useful to further explore the precise mechanism by which BACE1 and ␥-secretase function in cohort.Recently, our laboratory has been focusing on examining the role of voltage-gated sodium channel (VGSC) ␤ in the pathogenesis of Huntington disease and the regulation of APP processing in lipid rafts.2,3 VGSC is a large, multimeric complex that consists of an ␣ subunit and one or more ␤ subunits. To date, nine functional ␣ subunits and four ␤ subunits have been identified (11,12). Although VGSC␤ subunits are not essential to the basic operation of sodium channels, they are considered to be important auxiliary subunits, because co-expression of ␤ subunits are required to reconstitute full properties of the native sodium channel and to modify channel properties and intracellular localization (11,13). In the course of analyzing the VGSC␤, we found that these subunits are preferentially a...

Dravet syndrome (also called severe myoclonic epilepsy of infancy) is one of the most severe forms of childhood epilepsy. Most patients have heterozygous mutations in SCN1A, encoding voltage-gated sodium channel Na v 1.1 ␣ subunits. Sodium channels are modulated by ␤1 subunits, encoded by SCN1B, a gene also linked to epilepsy. Here we report the first patient with Dravet syndrome associated with a recessive mutation in SCN1B (p.R125C). Biochemical characterization of p.R125C in a heterologous system demonstrated little to no cell surface expression despite normal total cellular expression. This occurred regardless of coexpression of Na v 1.1 ␣ subunits. Because the patient was homozygous for the mutation, these data suggest a functional SCN1B null phenotype. To understand the consequences of the lack of ␤1 cell surface expression in vivo, hippocampal slice recordings were performed in Scn1b Ϫ/Ϫ versus Scn1b ϩ/ϩ mice. Scn1b Ϫ/Ϫ CA3 neurons fired evoked action potentials with a significantly higher peak voltage and significantly greater amplitude compared with wild type. However, in contrast to the Scn1a ϩ/Ϫ model of Dravet syndrome, we found no measurable differences in sodium current density in acutely dissociated CA3 hippocampal neurons. Whereas Scn1b Ϫ/Ϫ mice seize spontaneously, the seizure susceptibility of Scn1b ϩ/Ϫ mice was similar to wild type, suggesting that, like the parents of this patient, one functional SCN1B allele is sufficient for normal control of electrical excitability. We conclude that SCN1B p.R125C is an autosomal recessive cause of Dravet syndrome through functional gene inactivation.

Huntington's Disease (HD) is a dominantly inherited pathology caused by the accumulation of mutant huntingtin protein (HTT) containing an expanded polyglutamine (polyQ) tract. As the polyglutamine binding peptide 1 (QBP1) is known to bind an expanded polyQ tract but not the polyQ motif found in normal HTT, we selectively targeted mutant HTT for degradation by expressing a fusion molecule comprising two copies of QBP1 and copies of two different heat shock cognate protein 70 (HSC70)-binding motifs in cellular and mouse models of HD. Chaperone-mediated autophagy contributed to the specific degradation of mutant HTT in cultured cells expressing the construct. Intrastriatal delivery of a virus expressing the fusion molecule ameliorated the disease phenotype in the R6/2 mouse model of HD. Similar adaptor molecules comprising HSC70-binding motifs fused to an appropriate structure-specific binding agent(s) may have therapeutic potential for treating diseases caused by misfolded proteins other than those with expanded polyQ tracts.

Voltage-gated Na + channel (VGSC) β1 subunits regulate cell-cell adhesion and channel activity in vitro. We previously showed that β1 promotes neurite outgrowth in cerebellar granule neurons (CGNs) via homophilic cell adhesion, fyn kinase, and contactin. Here we demonstrate that β1-mediated neurite outgrowth requires Na + current (I Na ) mediated by Na v 1.6. In addition, β1 is required for highfrequency action potential firing. Transient I Na is unchanged in Scn1b (β1) null CGNs; however, the resurgent I Na , thought to underlie high-frequency firing in Na v 1.6-expressing cerebellar neurons, is reduced. The proportion of axon initial segments (AIS) expressing Na v 1.6 is reduced in Scn1b null cerebellar neurons. In place of Na v 1.6 at the AIS, we observed an increase in Na v 1.1, whereas Na v 1.2 was unchanged. This indicates that β1 is required for normal localization of Na v 1.6 at the AIS during the postnatal developmental switch to Na v 1.6-mediated high-frequency firing. In agreement with this, β1 is normally expressed with α subunits at the AIS of P14 CGNs. We propose reciprocity of function between β1 and Na v 1.6 such that β1-mediated neurite outgrowth requires Na v 1.6-mediated I Na , and Na v 1.6 localization and consequent high-frequency firing require β1. We conclude that VGSC subunits function in macromolecular signaling complexes regulating both neuronal excitability and migration during cerebellar development.V oltage-gated Na + channels (VGSCs), composed of one poreforming α subunit and two β subunits (1), are responsible for initiation and conduction of action potentials (APs) (2). Of the nine α subunits (3), Na v 1.1, Na v 1.2, and Na v 1.6 are found in the postnatal CNS (4, 5) where they display developmentally regulated expression patterns in specialized neuronal subcellular domains. For example, Na v 1.1 and Na v 1.2 are replaced during postnatal development at the axon initial segment (AIS) and nodes of Ranvier by Na v 1.6. Scn8a null mice display motor dysfunction, ataxia, and lethality by postnatal day (P) 21, suggesting that this developmental switch to Na v 1.6 expression in brain is critical (6, 7). Scn8a null retinal ganglion neurons display impaired excitability, demonstrating that Na v 1.6 is vital for high-frequency firing (8-10). Thus, VGSC-driven neuronal activity is important for proper CNS development, although the underlying mechanism(s) are not well understood.VGSC β1 subunits are multifunctional molecules that modulate channel kinetics and gating, regulate channel cell surface expression, and participate in cell-cell adhesion in vitro (11). Scn1b (β1) null mice are ataxic, experience spontaneous seizures, and exhibit a prolonged cardiac QT interval, demonstrating that β1 modulates electrical excitability in vivo (12, 13). Consistent with this, human mutations in SCN1B result in epilepsy and arrhythmia (14-21). As a member of the Ig superfamily of cell adhesion molecules (CAMs), β1 mediates cellular aggregation, cytoskeletal recruitment, and extracellular matrix interactio...

Pseudomonas aeruginosa, an opportunistic pathogen, is capable of establishing both chronic and acute infections in compromised hosts. Previous studies indicated that P. aeruginosa displays either a cytotoxic or an invasive phenotype in corneal epithelial cells. In this study, we used polarized MDCK cells for in vitro infection studies and confirmed that P. aeruginosa isolates can be broadly differentiated into two groups, expressing either a cytotoxic or an invasive phenotype. In vivo infection studies were performed to determine if cytotoxic and invasive strains displayed differential pathology. Invasion was assayed in vivo by in situ infection of mouse tracheal tissue followed by electron microscopy. Both cytotoxic and invasive strains entered mouse tracheal cells in situ; however, more necrosis was associated with the cytotoxic strain. In an acute lung infection model in rats, cytotoxic strains were found to damage lung epithelium more than invasive strains during the short infection period of this assay. The expression of cytotoxicity requires a functional exsA allele. In the strains tested, the ability to invade epithelial cells in vitro appears to be independent of exsA expression. Since ExsA is a transcriptional regulator of the exoenzyme S regulon, chromosomal preparations from invasive and cytotoxic strains were screened for their complement of exoenzyme S structural genes, exoS, encoding the 49-kDa ADP-ribosyltransferase (ExoS), and exoT, encoding the 53-kDa form of the enzyme (Exo53). Invasive strains possess both exoS and exoT, while cytotoxic strains appear to have lost exoS and retained exoT. These data indicate that the expression of cytotoxicity may be linked to the expression of Exo53, deletion of exoS and perhaps other linked loci, or expression of other ExsA-dependent virulence determinants. In the absence of a functional cytotoxicity pathway (exsA::⍀ strains), invasion of eukaryotic cells is detectable.