Over-expression of alpha-synuclein in the nervous system enhances axonal degeneration after peripheral nerve lesion in a transgenic mouse strain.

Wallerian degeneration in peripheral nerves occurs after a traumatic insult when the distal nerve part degenerates while peripheral macrophages enter the nerve stump and remove the accruing debris by phagozytosis. We used an experimental model to investigate the effect of either the absence or over-expression of alpha-synuclein (alpha-syn) after transecting the sciatic nerves of mice. alpha-Synuclein is a major component of Lewy bodies and its aggregation results in a premature destruction of nerve cells. It has also been found present in different peripheral nerves but its role in the axon remains still unclear. Following sciatic nerve transection in different mouse strains, we investigated the numbers of invading macrophages, the amounts of remaining myelin and axons 6 days after injury. All mice showed clear signs of Wallerian degeneration, but transgenic mice expressing human wild-type alpha-syn showed lower numbers of invading macrophages, less preserved myelin and significantly lower numbers of preserved axons in comparison with either knockout mice or a mouse strain with a spontaneous deletion of alpha-syn. The use of protein aggregation filtration blots and paraffin-embedded tissue blots displayed depositions of alpha-syn aggregates within sciatic nerve axons of transgenic mice. Thicker myelin sheaths and higher numbers of mitochondria were detected in old alpha-syn transgenic mice. In a human sural nerve, alpha-syn could also be identified within axons. Thus, alpha-syn and its aggregates are not only a component of Lewy bodies and synapses but also of axons and these aggregates might interfere with axonal transport. alpha-Synuclein transgenic mice represent an appropriate model for investigations on axonal transport in neurodegenerative diseases.

Localization of multiple acoustic sources with small arrays using a coherence test.

Direction finding of more sources than sensors is appealing in situations with small sensor arrays. Potential applications include surveillance, teleconferencing, and auditory scene analysis for hearing aids. A new technique for time-frequency-sparse sources, such as speech and vehicle sounds, uses a coherence test to identify low-rank time-frequency bins. These low-rank bins are processed in one of two ways: (1) narrowband spatial spectrum estimation at each bin followed by summation of directional spectra across time and frequency or (2) clustering low-rank covariance matrices, averaging covariance matrices within clusters, and narrowband spatial spectrum estimation of each cluster. Experimental results with omnidirectional microphones and colocated directional microphones demonstrate the algorithm's ability to localize 3-5 simultaneous speech sources over 4 s with 2-3 microphones to less than 1 degree of error, and the ability to localize simultaneously two moving military vehicles and small arms gunfire.

Selective detection, quantification, and subcellular location of alpha-synuclein aggregates with a protein aggregate filtration assay.

Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy are caused by alpha-synuclein aggregates. At present, there is no good biochemical method defining alpha-synuclein aggregates formed in vivo versus oligomers as a means to investigate alpha-synuclein aggregation and its mechanisms of neurodegeneration. A simple method, therefore, for the selective and sensitive detection of alpha-synuclein aggregates suited for screening purposes would be useful. Since in contrast to prions a proper detection of alpha-synuclein aggregates by Western blot analysis is difficult, we developed a protein aggregate filtration (PAF) assay. It takes advantage of the inherent insolubility of aggregated alpha-synuclein using microfiltration to separate it from soluble isoforms. For the first time, this assay even makes quantitative comparisons possible. We describe how the PAF assay can be applied to human brain tissue and animal and cell culture models, as well as used as a screening method for the subcellular location of alpha-synuclein aggregates. Since it detects the pathological isoform instead of surrogate markers, the PAF assay may have also potential in diagnosis of PD and DLB.

Presynaptic alpha-synuclein aggregates, not Lewy bodies, cause neurodegeneration in dementia with Lewy bodies.

Lewy bodies, the pathological hallmark of dementia with Lewy bodies (DLB), are large juxtanuclear inclusions of aggregated alpha-synuclein. However, the small number of cortical Lewy bodies relative to the total neuron count does not correlate with the extent of cognitive impairment. In contrast to dopaminergic neurons in Parkinson's disease, nerve cell loss is usually less prevalent in the cortex of DLB, suggesting a different mechanism of neurodegeneration. Because antibodies used for immunodetection per se do not generally differentiate the aggregated from the physiological and monomeric isoform of alpha-synuclein, we developed the paraffin-embedded tissue (PET) blot and the protein aggregate filtration (PAF) assay for the sensitive and selective detection of alpha-synuclein aggregates in tissue slides and brain homogenates, respectively. In contrast to common immunohistochemistry, the PET blot detected an enormous number of small alpha-synuclein aggregates, which, in contrast to the few Lewy bodies, may explain the cognitive impairment in DLB. Using the PAF assay, we demonstrate that the absolute majority of alpha-synuclein aggregates are located at presynaptic terminals, suggesting a severe pathological impact on synaptic function. Indeed, parallel to the massive presynaptic accumulation of alpha-synuclein aggregates, we observed significant synaptic pathology with almost complete loss of dendritic spines at the postsynaptic area. Our results provide strong evidence for a novel concept of neurodegeneration for DLB in which synaptic dysfunction is caused by presynaptic accumulation of alpha-synuclein aggregates. This concept may also be valid for Parkinson's disease.

Prion protein helix1 promotes aggregation but is not converted into beta-sheet.

Prion diseases are caused by the aggregation of the native alpha-helical prion protein PrP(C) into its pathological beta-sheet-rich isoform PrP(Sc). In current models of PrP(Sc), helix1 is assumed to be preferentially converted into beta-sheet during aggregation of PrP(C). This was supported by the NMR structure of PrP(C) since, in contrast to the isolated helix1, helix2 and helix3 are connected by a small loop and are additionally stabilized by an interhelical disulfide bond. However, helix1 is extremely hydrophilic and has a high helix propensity. This prompted us to investigate the role of helix1 in prion aggregation using humPrP(23-159) including helix1 (144-156) compared with the C-terminal-truncated isoform humPrP(23-144) corresponding to the pathological human stop mutations Q160Stop and Y145Stop, respectively. Most unexpectedly, humPrP(23-159) aggregated significantly faster compared with the truncated fragment humPrP(23-144), clearly demonstrating that helix1 is involved in the aggregation process. However, helix1 is not resistant to digestion with proteinase K in fibrillar humPrP(23-159), suggesting that helix1 is not converted to beta-sheet. This is confirmed by Fourier transformation infrared spectroscopy since there is almost no difference in beta-sheet content of humPrP(23-159) fibrils compared with humPrP(23-144). In conclusion, we provide strong direct evidence that in contrast to earlier assumptions helix1 is not converted into beta-sheet during aggregation of PrP(C) to PrP(Sc).

A new multiphasic buffer system for benzyldimethyl-n-hexadecylammonium chloride polyacrylamide gel electrophoresis of proteins providing efficient stacking.

Acidic PAGE systems using cationic detergents such as benzyldimethyl-n-hexadecylammonium chloride (16-BAC) or CTAB have proven useful for the detection of methoxy esters sensitive to alkaline pH, resolving basic proteins such as histones and membrane proteins. However, the interesting phosphate-based system suffered from poor stacking, resulting in broadened bands and long running times. Therefore, a new 16-BAC PAGE system based on the theory of moving boundary electrophoresis with properties comparable to the classical SDS-PAGE system was designed. As a result a new multiphasic analytical 16-BAC PAGE system providing efficient stacking and significantly shorter running times is presented here. It is based on acetic acid and methoxyacetic acid as common ion constituents. This PAGE system takes advantage of the additional counter stacking effect due to a cross boundary electrophoresis system resulting from the selected buffer constituents. Furthermore, the concentration of 16-BAC was optimized by determining its previously unknown CMC. Due to efficient focusing of the introduced tracking dye, methyl green, termination of electrophoresis can now be more easily followed as compared to the Schlieren line.

Modulation of L-type voltage-gated calcium channels by recombinant prion protein.

The prion protein (PrPC) has a primary role in the pathogenesis of transmissible spongiform encephalopathies. Here we analysed in detail the effect of recombinant PrPC and N- and C-terminal fragments of PrPC on the whole-cell current amplitude through voltage-gated calcium channels (VGCCs) of cultured wild-type cerebellar granule cells. With the application of full-length recombinant PrPC (50-500 nm), a highly significant reduction of the whole-cell current amplitude was observed in a dose-dependent manner. Amplitude reduction was abolished when cells were pre-incubated with nifedipine, a specific blocker of voltage-gated L-type calcium channels. N-terminal PrP fragments also led to a dose-dependent reduction of the maximal current amplitude, whereas a C-terminal fragment did not affect the current amplitude. These data demonstrate that nanomolar concentrations of PrPC modulate L-type VGCCs in mouse cerebellar granule cells, an effect that is dependent upon the copper-binding amino-terminal domain of PrPC.