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A mosaic of cross-phylum chemical interactions occurs between all metazoans and their microbiomes. A number of molecular families known to be produced by the microbiome have a profound impact on the balance between health and disease 1-9. Considering the diversity of the human microbiome, numbering over 40,000 operational taxonomic units 10 , the impact of the microbiome on the chemistry of an entire animal remains underexplored. In this study, mass spectrometry informatics and data visualization approaches 11-13 were used to provide an assessment of the impacts of the microbiome on the chemistry of an entire mammal by comparing metabolomics data from germ-free (GF) and specific pathogen Reprints and/or permissions can be provided by R. Quinn or P.C. Dorrestein.

␣-Synuclein (␣S) is a pre-synaptic protein that has been implicated as a possible causative agent in the pathogenesis of Parkinson's disease (PD). Two autosomal dominant missense mutations in the ␣S gene are associated with early onset PD. Because ␣S is found in an aggregated fibrillar form in the Lewy body deposits characteristic of Parkinson's patients, aggregation of the protein is believed to be related to its involvement in the disease process. The wild type (WT) and early onset mutants A30P and A53T display diverse in vitro aggregation kinetics even though the gross physicochemical and morphological properties of the mutants are highly similar. We used high resolution solution NMR spectroscopy to compare the structural and dynamic properties of the A53T and A30P mutants with those of WT ␣S in the free state. We found that the A30P mutation disrupts a region of residual helical structure that exists in the WT protein, whereas the A53T mutation results in a slight enhancement of a small region around the site of mutation with a preference for extended conformations. Based on these results and on the anticipated effects of these mutations on elements of secondary structure, we proposed a model of how these two PD-linked mutations influence ␣S fibril formation that is consistent with the documented differences in the fibrillization kinetics of the two mutants. ␣-Synuclein (␣S)1 is a 14.5-kDa amyloid fibril-forming protein strongly expressed in neurons that has been implicated in the pathogenesis of Parkinson's disease (PD) (1-4). Although the precise cellular function of ␣S remains obscure, a number of recent observations have highlighted its relevance to human pathology. Among those are genetic studies identifying several European kindred with heritable early onset PD that carry polymorphisms within the coding region of the ␣S gene resulting in one of two amino acid substitutions, A53T or A30P (5, 6). Further substantiation of the role of ␣S in PD arose from the finding that antibodies to ␣S epitopes decorate Lewy bodies extracted from affected brain regions of PD patients (7-10).Expression of human ␣S in transgenic animals produces PDlike symptoms and deposits (11, 12), whereas the ␣S double knockout mouse exhibits a subtle phenotype with no movement disorder (13). Comparison of these phenotypes suggests that A53T and A30P are gain of function mutations. Additional connections between ␣S and other neurological diseases continue to accumulate (4). For instance ␣S reportedly binds to and induces the aggregation of the amyloid-␤ peptide (A␤) of Alzheimer's disease (14, 15) and is also found in inclusions from dementia with Lewy bodies and multiple system atrophy (16). Thus, a preponderance of evidence suggests that ␣S plays a significant role in human neurological disease.The fact that aggregated ␣S is a major component of Lewy bodies in the brains of PD patients suggests that some aspect of ␣S aggregation may be relevant to PD pathology. The fibrillar morphology of aggregated ␣S for both the WT and the PDlinke...

The protein ␣-Synuclein (aS) is a synaptic vesicle-associated regulator of synaptic strength and dopamine homeostasis with a pathological role in Parkinson's disease. The normal function of aS depends on a membrane-associated conformation that is adopted upon binding to negatively charged lipid surfaces. Previously we found that the membrane-binding domain of aS is helical and suggested that it may exhibit an unusual structural periodicity. Here we present a study of the periodicity, topology, and dynamics of detergent micelle-bound aS using paramagnetic spin labels embedded in the micelle or attached to the protein. We show that the helical region of aS completes three full turns every 11 residues, demonstrating the proposed 11/3 periodicity. We also find that the membrane-binding domain is partially buried in the micelle surface and bends toward the hydrophobic interior, but does not traverse the micelle. Deeper submersion of certain regions within the micelle, including the unique lysine-free sixth 11-residue repeat, is observed and may be functionally important. There are no long-range tertiary contacts within this domain, indicating a highly extended configuration. The backbone dynamics of the micelle-bound region are relatively uniform with a slight decrease in flexibility observed toward the C-terminal end. These results clarify the topological features of aS bound to membrane-mimicking detergent micelles, with implications for aS function and pathology.Keywords: Synuclein; Parkinson's; amyloid; protein aggregation; membrane-associated proteins; helix periodicity ␣-Synuclein (aS) is a highly conserved presynaptic protein that plays a role in synaptic strength maintenance and dopamine homeostasis. Evidence that aS controls synaptic strength comes from neuronal cell line and knockout mouse models (Abeliovich et al. 2000;Murphy et al. 2000;Cabin et al. 2002;Schluter et al. 2003) that display impaired synaptic response to repetitive stimuli and alterations in the number of reserve pool vesicles, suggesting that aS regulates reserve synaptic vesicles called upon when readily releasable vesicles are exhausted. aS may accomplish this in part by interacting with and regulating phospholipase D (PLD) (Jenco et al. 1998;Ahn et al. 2002;Outeiro and Lindquist 2003;Payton et al. 2004), an enzyme with a purported role in vesicular trafficking (Liscovitch et al. 2000). aS also appears to be involved in regulating intracellular dopamine levels at several points of control. Expression of aS alters synaptic membrane permeability to dopamine by interacting with the human dopamine transporter (hDAT), resulting in withdrawal of hDAT from the external membrane . aS can also block dopamine synthesis by inhibiting tyrosine hydroxylase (Perez et al. 2002). In addition, aS can influence the activity of the vesicular dopamine transporter VMAT2 (Lotharius et al. 2002). Article published online ahead of print. Article and publication date are at

Alpha-synuclein (alphaS) is a lipid-binding synaptic protein of unknown function that is found in an aggregated amyloid fibril form in the intraneuronal Lewy body deposits that are a defining characteristic of Parkinson's disease (PD). Although intrinsically unstructured when free in solution, alphaS adopts a highly helical conformation in association with lipid membranes or membrane mimetic detergent micelles. Two mutations in the alphaS gene have been linked to early onset autosomal dominant hereditary forms of PD, and have been shown to affect the aggregation kinetics of the protein in vitro. We have used high-resolution NMR spectroscopy, circular dichroism, and limited proteolysis to investigate the effects of these PD-linked mutations on the helical structure adopted by alphaS in the lipid or detergent micelle-bound form. We show that neither the A53T nor the A30P mutation has a significant effect on the structure of the folded protein, although the A30P mutation may cause a minor perturbation in the helical structure around the site of the mutation. The A30P, but not the A53T, mutation also appears to decrease the affinity of the protein for lipid surfaces, possibly by perturbing the nascent helical structure of the free protein. The potential implications of these results for the role of alphaS in PD are discussed.

Background.-Functional Magnetic Resonance Imaging (fMRI) in awake behaving mice is well positioned to bridge the detailed cellular-level view of brain activity, which has become available due to recent advances in microscopic optical imaging and genetics, to the macroscopic scale of human noninvasive observables. However, while microscopic (e.g., 2-photon imaging) studies in behaving mice have become a reality in many laboratories, awake mouse fMRI remains a

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