Nobuyuki Nukina

Visualization of Aβ42(43) and Aβ40 in senile plaques with end-specific Aβ monoclonals: Evidence that an initially deposited species is Aβ42(43)

To learn about the carboxy-terminal extent of amyloid beta-protein (A beta) composition of senile plaques (SPs) in the brain affected with Alzheimers disease (AD), we employed two end-specific monoclonal antibodies as immunocytochemical probes: one is specific for A beta 40, the carboxyl terminus of A beta 1-40, while the other is specific for A beta 42(43). In the AD cortex, all SPs that were labeled with an authentic antibody were A beta 42(43) positive, while only one-third of which, on the average, were A beta 40 positive. There was a strong correlation between A beta 40 positivity and mature plaques. Two familial AD cortices with the mutation of beta-amyloid protein precursor 717 (beta APP717) (Val to Ile) showed a remarkable predominance of A beta 42(43)-positive, A beta 40-negative plaques. Diffuse plaques, representing the earliest stage of A beta deposition, were exclusively positive for A beta 42(43), but completely negative for A beta 40.

Trehalose alleviates polyglutamine-mediated pathology in a mouse model of Huntington disease

Inhibition of polyglutamine-induced protein aggregation could provide treatment options for polyglutamine diseases such as Huntington disease. Here we showed through in vitro screening studies that various disaccharides can inhibit polyglutamine-mediated protein aggregation. We also found that various disaccharides reduced polyglutamine aggregates and increased survival in a cellular model of Huntington disease. Oral administration of trehalose, the most effective of these disaccharides, decreased polyglutamine aggregates in cerebrum and liver, improved motor dysfunction and extended lifespan in a transgenic mouse model of Huntington disease. We suggest that these beneficial effects are the result of trehalose binding to expanded polyglutamines and stabilizing the partially unfolded polyglutamine-containing protein. Lack of toxicity and high solubility, coupled with efficacy upon oral administration, make trehalose promising as a therapeutic drug or lead compound for the treatment of polyglutamine diseases. The saccharide-polyglutamine interaction identified here thus provides a new therapeutic strategy for polyglutamine diseases.

Eukaryotic Proteasomes Cannot Digest Polyglutamine Sequences and Release Them during Degradation of Polyglutamine-Containing Proteins

Long glutamine sequences (polyQ) occur in many cell proteins, and several neurodegenerative diseases result from expansion of these sequences. PolyQ-containing proteins are degraded by proteasomes, whose three active sites prefer to cleave after hydrophobic, basic, or acidic residues. We tested whether these particles can digest a polyQ chain. Eukaryotic 26S and 20S proteasomes failed to cut within stretches of 9-29Q residues in peptides. While digesting a myoglobin Q(35) fusion protein, the proteasomes spared the polyQ sequence. In contrast, archaeal proteasomes, whose 14 active sites are less specific, rapidly digested such polyQ repeats. Therefore, when degrading polyQ proteins, eukaryotic proteasomes must release aggregation-prone polyQ-containing fragments for further hydrolysis by unidentified peptidases. In polyQ diseases, such polyQ sequences (38-300Qs) exceed the lengths of normal proteasome products (2-25 residues). Occasional failure of these long undegradable sequences to exit may interfere with proteasome function and help explain why longer polyQ expansions promote early disease onset.

Chromogranin-mediated secretion of mutant superoxide dismutase proteins linked to amyotrophic lateral sclerosis.

Here we report that chromogranins, components of neurosecretory vesicles, interact with mutant forms of superoxide dismutase (SOD1) that are linked to amyotrophic lateral sclerosis (ALS), but not with wild-type SOD1. This interaction was confirmed by yeast two-hybrid screen and by co-immunoprecipitation assays using either lysates from Neuro2a cells coexpressing chromogranins and SOD1 mutants or lysates from spinal cord of ALS mice. Confocal and immunoelectron microscopy revealed a partial colocalization of mutant SOD1 with chromogranins in spinal cord of ALS mice. Mutant SOD1 was also found in immuno-isolated trans-Golgi network and in microsome preparations, suggesting that it can be secreted. Indeed we report evidence that chromogranins may act as chaperone-like proteins to promote secretion of SOD1 mutants. From these results, and our finding that extracellular mutant SOD1 can trigger microgliosis and neuronal death, we propose a new ALS pathogenic model based on the toxicity of secreted SOD1 mutants.

Semi‐rational engineering of a coral fluorescent protein into an efficient highlighter

Kaede is a natural photoconvertible fluorescent protein found in the coral Trachyphyllia geoffroyi. It contains a tripeptide, His 62‐Tyr 63‐Gly 64, which acts as a green chromophore that is photoconvertible to red following (ultra‐) violet irradiation. Here, we report the molecular cloning and crystal structure determination of a new fluorescent protein, KikG, from the coral Favia favus, and its in vitro evolution conferring green‐to‐red photoconvertibility. Substitution of the His 62‐Tyr 63‐Gly 64 sequence into the native protein provided only negligible photoconversion. On the basis of the crystal structure, semi‐rational mutagenesis of the amino acids surrounding the chromophore was performed, leading to the generation of an efficient highlighter, KikGR. Within mammalian cells, KikGR is more efficiently photoconverted and is several‐fold brighter in both the green and red states than Kaede. In addition, KikGR was successfully photoconverted using two‐photon excitation microscopy at 760 nm, ensuring optical cell labelling with better spatial discrimination in thick and highly scattering tissues.

beta Subunits of voltage-gated sodium channels are novel substrates of beta-site amyloid precursor protein-cleaving enzyme (BACE1) and gamma-secretase

Sequential processing of amyloid precursor protein (APP) by membrane-bound proteases, BACE1 and γ-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 β subunits of voltage-gated sodium channels are sequentially processed by BACE1 and γ-secretase. These results may provide new insights into the underlying pathology of Alzheimer disease.

Deranged Calcium Signaling and Neurodegeneration in Spinocerebellar Ataxia Type 3

Spinocerebellar ataxia type 3 (SCA3), also known as Machado–Joseph disease (MJD), is an autosomal-dominant neurodegenerative disorder caused by a polyglutamine expansion in ataxin-3 (ATX3; MJD1) protein. In biochemical experiments, we demonstrate that mutant ATX3exp specifically associated with the type 1 inositol 1,4,5-trisphosphate receptor (InsP3R1), an intracellular calcium (Ca2+) release channel. In electrophysiological and Ca2+ imaging experiments, we show that InsP3R1 was sensitized to activation by InsP3 in the presence of mutant ATX3exp. We found that feeding SCA3-YAC-84Q transgenic mice with dantrolene, a clinically relevant stabilizer of intracellular Ca2+ signaling, improved their motor performance and prevented neuronal cell loss in pontine nuclei and substantia nigra regions. Our results indicate that deranged Ca2+ signaling may play an important role in SCA3 pathology and that Ca2+ signaling stabilizers such as dantrolene may be considered as potential therapeutic drugs for treatment of SCA3 patients.

Huntington's disease intranuclear inclusions contain truncated, ubiquitinated huntingtin protein.

Intranuclear inclusion bodies are a shared pathological feature of Huntingtons disease (HD) and its transgenic mouse model. Using a panel of antibodies spanning the entire huntingtin molecule, we have investigated the pattern of immunoreactivity within the intranuclear inclusions in the frontal cortex and striatum of patients with HD. The intranuclear inclusions reacted with anti-ubiquitin and antibodies against the N-terminal portion of huntingtin (CAG53b, HP1), but not with HD1 and the 1C2 antibodies that detect the expanded polyglutamine tract nor the more C-terminal antibodies. However, the 1C2, HP1, CAG53b, and HD1 antibodies detected granular cytoplasmic deposits in cortical and striatal neurons that also contained intranuclear N-terminal huntingtin immunoreactivity. These data show a differential intracellular location of truncated huntingtin in the HD brain. Both the cytoplasmic and the nuclear aggregates of the protein fragments could be neurotoxic. The frequency of the cortical intranuclear inclusions correlated with the size of CAG expansion and was inversely related to the age at onset and death. No such correlations were detected for the striatum, which most likely reflects a more advanced neuronal loss accrued by the time of death.

A Functional Null Mutation of SCN1B in a Patient with Dravet Syndrome

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 Nav1.1 α subunits. Sodium channels are modulated by β1 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 Nav1.1 α 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 β1 cell surface expression in vivo, hippocampal slice recordings were performed in Scn1b−/− versus Scn1b+/+ mice. Scn1b−/− 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+/− model of Dravet syndrome, we found no measurable differences in sodium current density in acutely dissociated CA3 hippocampal neurons. Whereas Scn1b−/− mice seize spontaneously, the seizure susceptibility of Scn1b+/− 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.

Distinct conformations of in vitro and in vivo amyloids of huntingtin-exon1 show different cytotoxicity.

A hallmark of polyglutamine diseases, including Huntington disease (HD), is the formation of β-sheet-rich aggregates, called amyloid, of causative proteins with expanded polyglutamines. However, it has remained unclear whether the polyglutamine amyloid is a direct cause or simply a secondary manifestation of the pathology. Here we show that huntingtin-exon1 (thtt) with expanded polyglutamines remarkably misfolds into distinct amyloid conformations under different temperatures, such as 4 °C and 37 °C. The 4 °C amyloid has loop/turn structures together with mostly β-sheets, including exposed polyglutamines, whereas the 37 °C amyloid has more extended and buried β-sheets. By developing a method to efficiently introduce amyloid into mammalian cells, we found that the formation of the 4 °C amyloid led to substantial toxicity, whereas the toxic effects of the 37 °C amyloid were very small. Importantly, thtt amyloids in different brain regions of HD mice also had distinct conformations. The thermolabile thtt amyloid with loop/turn structures in the striatum showed higher toxicity, whereas the rigid thtt amyloid with more extended β-sheets in the hippocampus and cerebellum had only mild toxic effects. These studies show that the thtt protein with expanded polyglutamines can misfold into distinct amyloid conformations and, depending on the conformations, the amyloids can be either toxic or nontoxic. Thus, the amyloid conformation of thtt may be a critical determinant of cytotoxicity in HD.