Familial amyotrophic lateral sclerosis-linked mutant SOD1 aberrantly interacts with tubulin.

Mutations in the Cu,Zn-superoxide dismutase (SOD1) gene cause 20-25% of familial amyotrophic lateral sclerosis (ALS). Mutant SOD1 causes motor neuron degeneration through toxic gain-of-function(s). However, the direct molecular targets of mutant SOD1, underlying its toxicity, are not fully understood. In this study, we found that alpha/beta-tubulin is one of the major mutant SOD1-interacting proteins, but that wild-type SOD1 does not interact with it. The interaction between tubulin and mutant SOD1 was detected in the spinal cords of mutant G93A SOD1 transgenic mice before the onset of symptoms. Tubulin interacted with amino acid residues 1-23 and 116-153 of SOD1. Overexpression of mutant SOD1 resulted in the accumulation of tubulin in detergent-insoluble fractions. In a cell-free system, mutant SOD1 modulated tubulin polymerization, while wild-type SOD1 did not. Since tightly regulated microtubule dynamics is essential for neurons to remain viable, alpha/beta-tubulin could be an important direct target of mutant SOD1.

Effective amino acid composition of seaweeds inducing food preference behaviors in Aplysia kurodai.

Aplysia kurodai feeds on Ulva but rejects Gelidium and Pachydictyon with distinct patterned jaw movements. We previously demonstrated that these movements are induced by taste alone. Thus some chemicals may contribute to induction of these responses. We explored the amino acids composition of Ulva, Gelidium and Pachydictyon extracts used during our taste-induced physiological experiments. These solutions contained many constituents. The concentrations of six amino acids (Asp, Asn, Glu, Gln, Phe, Tau) were obviously different in the three extract solutions. We explored patterned jaw movements following application of solutions containing a pure amino acid. We statistically compared the occurrence numbers of ingestion-like and rejection-like patterned jaw movements (positive and negative values, respectively) for each amino acid. Our results suggested that L-Asn tends to induce ingestion-like responses, likely resulting in a preference of Ulva. In contrast, L-Asp tends to induce rejection-like responses, likely resulting in aversion towards Pachydictyon. In addition, we demonstrated that L-Asn and L-Asp solutions were sufficient to induce muscle activity associated with ingestion-like or rejection-like responses in the jaw muscles of a semi-intact preparation.

Aberrant molecular properties shared by familial Parkinson's disease-associated mutant UCH-L1 and carbonyl-modified UCH-L1.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by loss of dopaminergic neurons. The I93M mutation in ubiquitin C-terminal hydrolase L1 (UCH-L1) is associated with familial PD, and we have previously shown that the I93M UCH-L1-transgenic mice exhibit dopaminergic cell loss. Over 90% of neurodegenerative diseases, including PD, occur sporadically. However, the molecular mechanisms underlying sporadic PD as well as PD associated with I93M UCH-L1 are largely unknown. UCH-L1 is abundant (1-5% of total soluble protein) in the brain and is a major target of oxidative/carbonyl damage associated with sporadic PD. As well, abnormal microtubule dynamics and tubulin polymerization are associated with several neurodegenerative diseases including frontotemporal dementia and parkinsonism linked to chromosome 17. Here we show that familial PD-associated mutant UCH-L1 and carbonyl-modified UCH-L1 display shared aberrant properties: compared with wild-type UCH-L1, they exhibit increased insolubility and elevated interactions with multiple proteins, which are characteristics of several neurodegenerative diseases-linked mutants. Circular dichroism analyses suggest similar structural changes in both UCH-L1 variants. We further report that one of the proteins interacting with UCH-L1 is tubulin, and that aberrant interaction of mutant or carbonyl-modified UCH-L1 with tubulin modulates tubulin polymerization. These findings may underlie the toxic gain of function by mutant UCH-L1 in familial PD. Our results also suggest that the carbonyl modification of UCH-L1 and subsequent abnormal interactions of carbonyl-modified UCH-L1 with multiple proteins, including tubulin, constitute one of the causes of sporadic PD.

A CPG component LE generates depolarization of buccal neurons by producing constant plateau potentials during feeding responses of Aplysia kurodai.

In the buccal ganglia of Aplysia kurodai we have identified neurons (here termed LE neurons, or LE) producing plateau potentials lasting several seconds by application of short depolarizing currents. Results obtained from experiments using various bath solutions suggest that generation of these plateau potentials may be an endogenous property of LE. Application of various intensities or lengths of depolarizing currents induced in LE almost constant plateau potentials with fixed duration and depolarizing size. LE spikes produced monosynaptic EPSPs in the ipsilateral multi-action neuron (MA) and the jaw-closing motor neuron (JC) in the buccal ganglia. Conversely, MA spikes produced monosynaptic IPSPs in LE. There was electrical coupling between LE and both MA and JC. During the feeding-like response elicited by electrical stimulation of the nerve, LE showed rhythmic depolarization almost simultaneously with MA and JC, and firing on the plateau potentials occurred during the period of JC firing, the later phase of radula retraction. Hyperpolarization of LE during the feeding-like response suppressed generation of plateau potentials, though rhythmic small depolarization was still induced. During LE hyperpolarization, the duration of the depolarization of MA and JC was shortened. These results suggest that LE may be an element of the feeding CPG circuit and may contribute to part of the depolarization of MA and JC by generating constant plateau potentials during the feeding response, though LE may not have rhythm-generating ability.

Responses of cerebral GABA-containing CBM neuron to taste stimulation with seaweed extracts in Aplysia kurodai.

Aplysia kurodai distributed along Japan feeds well on Ulva pertusa but rejects Gelidium amansii with distinctive patterned movements of the jaws and radula. On the ventral side of the cerebral M cluster, four cell bodies of higher order neurons that send axons to the buccal ganglia are distributed (CBM neurons). We have previously shown that the dopaminergic CBM1 modulates basic feeding circuits in the buccal ganglia for rejection by firing at higher frequency after application of the aversive taste of seaweed such as Gelidium amansii. In the present experiments immunohistochemical techniques showed that the CBM3 exhibited gamma-aminobutyric acid (GABA)-like immunoreactivity. The CBM3 may be equivalent to the CBI-3 involved in changing the motor programs from rejection to ingestion in Aplysia californica. The responses of the CBM3 to taste stimulation of the lips with seaweed extracts were investigated by the use of calcium imaging. The calcium-sensitive dye, Calcium Green-1, was iontophoretically introduced into a cell body of the CBM3 using a microelectrode. Application of Ulva pertusa or Gelidium amansii extract induced different changes in fluorescence in the CBM3 cell body, indicating that taste of Ulva pertusa initially induced longer-lasting continuous spike responses at slightly higher frequency compared with that of Gelidium amansii. Considering a role of the CBM3 in the pattern selection, these results suggest that elongation of the initial firing response may be a major factor for the CBM3 to switch the buccal motor programs from rejection to ingestion after application of different tastes of seaweeds in Aplysia kurodai.