High Accuracy Ion Mobility Spectrometry for Instrument Calibration.

Ion mobility spectrometry (IMS) is widely used to characterize compounds of interest (COIs) based on their reduced mobility ( K0) values. In an attempt to increase the accuracy and agreement of studies, the most recommended method has been to use a reference compound with a known K0 value to calibrate the instrument and calculate COI K0 values from normalized spectra. Researchers are limited by the accuracy of previous K0 value reference measurements on which to base their calibrations. Any inaccuracy in these reference K0 values, typically ±2%, will propagate through to the calculated K0 value of the COI. For this reason, there is a need to standardize reference K0 values with improved accuracy. Through improvement of the accuracy of reference measurements, a lower degree of error will propagate through new K0 value calculations. The K0 values of the ammonium reactant ion, the potential reference standard dimethyl methylphosphonate (DMMP), and three explosive COIs were characterized at multiple drift gas temperatures, drift gas water contents, and electric field strengths on an accurate ion mobility spectrometry instrument. K0 values reported here are known to ±0.1% as a result of reducing the error of all instrumental parameters.

Ambient Pressure Inverse Ion Mobility Spectrometry Coupled to Mass Spectrometry.

Although higher resolving powers are often achieved using ambient pressure drift tube ion mobility mass spectrometry (DT-IMMS) systems, lower duty cycles are often required which directly impacts sensitivity. Moreover, the mechanism of ion gating using Bradbury-Nielsen or Tyndall-Gate configurations routinely results in ion gate depletion effects which discriminate against low mobility ions. This paper reports a new method of ambient pressure ion mobility operation in which inverse ion mobility spectrometry is coupled to a time-of-flight mass spectrometer to improve sensitivity and minimize the effects of ion gate depletion. In this mode of operation, the duty cycle is improved to approximate 99% from a typical value of less than 1%, improving the signal intensity by over 2 orders of magnitude. Another advantage of inverse ion mobility mass spectrometry is a reduction of the impact of ion gate depletion on low mobility molecules that translates into higher sensitivity for this class of analytes. To demonstrate these benefits afforded by this instrumental mode of operation differences in sensitivity, resolving power, and ion discrimination are compared between the inverse and normal modes of operation using tetraalkylammonium standards. These results show that the ion throughput is significantly increased for analytes with a broad range of mobilities with little impact on resolving power. While the mobility-based discrimination is minimized using the inverse mode of operation, the noise level in the inverse mode is highly dependent upon the stability of ionization source.

Determination of E/N Influence on K0 Values within the Low Field Region of Ion Mobility Spectrometry.

The established theory of ion motion within weak electric fields predicts that reduced ion mobility (K0) remains constant as a function of the ratio of electric field strength to drift gas number density (E/N). However, upon increasing the accuracy and precision of K0 value measurements during a previous study, a new relationship was seen in which the K0 values of ions decreased as a function of increasing E/N at field strengths below 4 Td. Here the effect of E/N on the K0 value of an ion has been investigated in order to validate the reality of the phenomenon and determine its cause. The pertinent measurements of voltage and drift time were verified in order to ensure the authenticity of the trend and that it was not a result of a systematic error in parametric measurements. The trend was also replicated on a separate ion mobility spectrometer drift tube in order to further validate its authenticity. As a result, the theory of ion motion within weak electric fields should be revised to reflect the behavior seen here.

Fecal Metabolome in Hmga1 Transgenic Mice with Polyposis: Evidence for Potential Screen for Early Detection of Precursor Lesions in Colorectal Cancer.

Because colorectal cancer (CRC) remains a leading cause of cancer mortality worldwide, more accessible screening tests are urgently needed to identify early stage lesions. We hypothesized that highly sensitive, metabolic profile analysis of stool samples will identify metabolites associated with early stage lesions and could serve as a noninvasive screening test. We therefore applied traveling wave ion mobility mass spectrometry (TWIMMS) coupled with ultraperformance liquid chromatography (UPLC) to investigate metabolic aberrations in stool samples in a transgenic model of premalignant polyposis aberrantly expressing the gene encoding the high mobility group A (Hmga1) chromatin remodeling protein. Here, we report for the first time that the fecal metabolome of Hmga1 mice is distinct from that of control mice and includes metabolites previously identified in human CRC. Significant alterations were observed in fatty acid metabolites and metabolites associated with bile acids (hypoxanthine xanthine, taurine) in Hmga1 mice compared to controls. Surprisingly, a marked increase in the levels of distinctive short, arginine-enriched, tetra-peptide fragments was observed in the transgenic mice. Together these findings suggest that specific metabolites are associated with Hmga1-induced polyposis and abnormal proliferation in intestinal epithelium. Although further studies are needed, these data provide a compelling rationale to develop fecal metabolomic analysis as a noninvasive screening tool to detect early precursor lesions to CRC in humans.

Correcting the fundamental ion mobility equation for field effects.

The fundamental ion mobility equation computes the energy-averaged collision cross section as a function of measured drift velocity, electric field strength, ion and neutral masses, and drift gas state parameters. As field strength approaches zero, in particular when the drift velocity drops below about 4% of the average ion-neutral thermal speed, the fundamental equation takes on an especially simple form because the collision frequency and average momentum transfer become indistinguishable from their thermal values. However, in modern high-performance IMS-MS instruments, ion drift velocities may be 10-50% or more of thermal speed, and analysis using the zero-field equation gives rise to erroneously large cross sections. We address this problem by developing correction factors for the zero-field equation from an improved momentum-transfer (MT) theory for ion mobility, corrected and completed herein, and from the well-known two-temperature (2T) theory. The corrected and uncorrected equations are compared by their ability to recover known hard-sphere cross sections from accurately-computed mobility data. Both MT and 2T expressions adjust for the field-driven increase in collision frequency and are noticeably superior to zero-field expression whenever the ion drift velocity is greater than ∼4% of thermal speed. The MT expression also adjusts for the mass and field dependent change in average momentum transfer, and is more accurate than the 2T first approximation whenever the mass of the ionic species is greater than about four times that of drift gas molecules, as is the case in most analytical applications of IMS coupled to MS.

Neuronal metabolomics by ion mobility mass spectrometry in cocaine self-administering rats after early and late withdrawal.

The neuronal metabolomes in rat striatum (STR), prefrontal cortex (PFC), and nucleus accumbens (NAC) were analyzed by Hadamard transform ion mobility mass spectrometry (HT-IMMS) in order to reveal global and specific metabolic changes induced by cocaine self-administration after 1-day or 3-week withdrawal. Metabolite features were comprehensively separated and detected using HPLC-IMMS within minutes. Global metabolic differences were observed by PCA for comparisons between cocaine and saline treatments at 1-day withdrawal time. Metabolite features that were significantly changed were selected using PCA loadings' plot and unpaired LLL test and then tentatively identified by accurate m/z, yielding a complete profile of metabolic changes induced by cocaine self-administration. The majority of these changes were found at the 1-day withdrawal time, but several of them endured even after 3-week withdrawal from cocaine, and these changes were generally brain region specific. Putatively identified metabolites associated with oxidative stress and energy metabolism were also specifically investigated. We discovered that the dysregulation of creatine/creatinine was different between the STR and NAC, demonstrating that metabolic alterations are brain region specific. Glutathione and adenosine were also changed in their abundance, and the results agreed with previous studies. In general, this study provided a high-throughput analytical platform to perform metabolomics analyses with putative identifications for altered metabolite features induced by cocaine treatment, therefore revealing additional metabolic targets of cocaine-induced changes after early and extended withdrawal times.

HMGA1 drives metabolic reprogramming of intestinal epithelium during hyperproliferation, polyposis, and colorectal carcinogenesis.

Although significant progress has been made in the diagnosis and treatment of colorectal cancer (CRC), it remains a leading cause of cancer death worldwide. Early identification and removal of polyps that may progress to overt CRC is the cornerstone of CRC prevention. Expression of the High Mobility Group A1 (HMGA1) gene is significantly elevated in CRCs as compared with adjacent, nonmalignant tissues. We investigated metabolic aberrations induced by HMGA1 overexpression in small intestinal and colonic epithelium using traveling wave ion mobility mass spectrometry (TWIMMS) in a transgenic model in which murine Hmga1 was misexpressed in colonic epithelium. To determine if these Hmga1-induced metabolic alterations in mice were relevant to human colorectal carcinogenesis, we also investigated tumors from patients with CRC and matched, adjacent, nonmalignant tissues. Multivariate statistical methods and manual comparisons were used to identify metabolites specific to Hmga1 and CRC. Statistical modeling of data revealed distinct metabolic patterns in Hmga1 transgenics and human CRC samples as compared with the control tissues. We discovered that 13 metabolites were specific for Hmga1 in murine intestinal epithelium and also found in human CRC. Several of these metabolites function in fatty acid metabolism and membrane composition. Although further validation is needed, our results suggest that high levels of HMGA1 protein drive metabolic alterations that contribute to CRC pathogenesis through fatty acid synthesis. These metabolites could serve as potential biomarkers or therapeutic targets.

Determining the isomeric heterogeneity of neutral oligosaccharide-alditols of bovine submaxillary mucin using negative ion traveling wave ion mobility mass spectrometry.

Negative ions produced by electrospray ionization were used to evaluate the isomeric heterogeneity of neutral oligosaccharide-alditols isolated from bovine submaxillary mucin (BSM). The oligosaccharide-alditol mixture was preseparated on an off-line high-performance liquid chromatography (HPLC) column, and the structural homogeneity of individual LC fractions was investigated using a Synapt G2 traveling wave ion mobility spectrometer coupled between quadupole and time-of-flight mass spectrometers. Mixtures of isomers separated by both chromatography and ion mobility spectrometry were studied. Tandem mass spectrometry (MS/MS) of multiple mobility peaks having the same mass-to-charge ratio (m/z) demonstrated the presence of different structural isomers and not differences in ion conformations due to charge site location. Although the oligosaccharide-alditol mixture was originally separated by HPLC, multiple ion mobility peaks due to structural isomers were observed for a number of oligosaccharide-alditols from single LC fractions. The collision-induced dissociation cells located in front of and after the ion mobility separation device enabled oligosaccharide precursor or product ions to be separated by ion mobility and independent fragmentation spectra to be acquired for isomeric carbohydrate precursor or product ions. MS/MS spectra so obtained for independent mobility peaks at a single m/z demonstrated the presence of structural variants or stereochemical isomers having the same molecular formula. This was observed both for oligosaccharide precursor and product ions. In addition, mobilities of both [M - H](-) and [M + Cl](-) ions, formed by adding NH4OH or NH4Cl to the electrospray solvent, were examined and compared for selected oligosaccharide-alditols. Better separation among structural isomers appeared to be achieved for some [M + Cl](-) anions.

Characterizing metabolic changes in human colorectal cancer.

Colorectal cancer (CRC) remains a leading cause of cancer death worldwide, despite the fact that it is a curable disease when diagnosed early. The development of new screening methods to aid in early diagnosis or identify precursor lesions at risk for progressing to CRC will be vital to improving the survival rate of individuals predisposed to CRC. Metabolomics is an advancing area that has recently seen numerous applications to the field of cancer research. Altered metabolism has been studied for many years as a means to understand and characterize cancer. However, further work is required to establish standard procedures and improve our ability to identify distinct metabolomic profiles that can be used to diagnose CRC or predict disease progression. The present study demonstrates the use of direct infusion traveling wave ion mobility mass spectrometry to distinguish metabolic profiles from CRC samples and matched non-neoplastic epithelium as well as metastatic and primary tumors at different stages of disease (T1-T4). By directly infusing our samples, the analysis time was reduced significantly, thus increasing the speed and efficiency of this method compared to traditional metabolomics platforms. Partial least squares discriminant analysis was used to visualize differences between the metabolic profiles of sample types and to identify the specific m/z features that led to this differentiation. Identification of the distinct m/z features was made using the human metabolome database. We discovered alterations in fatty acid biosynthesis and oxidative, glycolytic, and polyamine pathways that distinguish tumors from non-malignant colonic epithelium as well as various stages of CRC. Although further studies are needed, our results indicate that colonic epithelial cells undergo metabolic reprogramming during their evolution to CRC, and the distinct metabolites could serve as diagnostic tools or potential targets in therapy or primary prevention. Graphical Abstract Colon tissue biopsy samples were collected from patients after which metabolites were extracted via sonication. Two-dimensional data were collected via IMS in tandem with MS (IMMS). Data were then interpreted statistically via PLS-DA. Scores plots provided a visualization of statistical separation and groupings of sample types. Loading plots allowed identification of influential ion features. Lists of these features were exported and analyzed for specific differences. Direct comparisons of the ion features led to the identification and comparative analyses of candidate biomarkers. These differences were then expressed visually in charts and tables.

Metabolic analysis of striatal tissues from Parkinson's disease-like rats by electrospray ionization ion mobility mass spectrometry.

Electrospray ionization ion mobility mass spectrometry (ESI-IMMS) was used to study the striatal metabolomes in a Parkinson's like disease (PD-like) rat model. Striatal tissue samples from Berlin Druckrey IV (BD-IV) with PD-like disease 20 dpn-affected and 15 dpn-affected rats (dpn: days postnatal) were investigated and compared with age-matched controls. An ion mobility mass spectrometer (IMMS) produced multidimensional spectra with mass to charge ratio (m/z), ion mobility drift time, and intensity information for each individual metabolite. Principle component analysis (PCA) was applied in this study for pattern recognition and significant metabolites selection (68% data was modeled in PCA). Both IMMS spectra and PCA results showed that there were clear global metabolic differences between PD-like samples and healthy controls. Nine metabolites were selected by PCA and identified as potential biomarkers using the Human Metabolome Database (HMDB). One targeted metabolite in this study was dopamine. Selected-mass mobility analysis indicated the absence of dopamine in PD-like striatal metabolomes. A major discovery of this work, however, was the existence of an isomer of dopamine. By using ion mobility spectrometry, the dopamine isomer, which has not previously been reported, was separated from dopamine.

Evaluation of Hadamard transform atmospheric pressure ion mobility time-of-flight mass spectrometry for complex mixture analysis.

Ion mobility mass spectrometry (IMMS) has gained popularity in the analysis of complex mixtures such as those encountered in metabolomics and proteomics. However, the challenge that exists in conventional pulsed IMMS is its inherent low duty cycle. The first application of Hadamard transform (HT)-type signal coupled with atmospheric pressure IMMS to complex mixtures is presented. Performance of the prototype was assessed by the analysis of metabolite standard mixture. With 200 times increased IMS duty cycle in HT mode compared with conventional pulsed mode, the limit of detection (LOD) was decreased by ∼10 times. Evaluation for application to complex mixtures was achieved using the NIST Standard Reference Material 1950 Metabolites in Human Plasma. Approximately 180 metabolite ions were detected within 1 min with an IMS resolving power (Rp) of ∼100. Rapid chromatographic separation prior to IMMS analysis was also demonstrated for improving the response of metabolite ions in rat brain tissue extract.

Desorption electrospray ionization (DESI) with atmospheric pressure ion mobility spectrometry for drug detection.

Desorption electrospray ionization (DESI) was coupled to an ambient pressure drift tube ion mobility time-of-flight mass spectrometer (IM-TOFMS) for the direct analysis of active ingredients in pharmaceutical samples. The DESI source was also coupled with a standalone IMS demonstrating potential of portable and inexpensive drug-quality testing platforms. The DESI-IMS required no sample pretreatment as ions were generated directly from tablets and cream formulations. The analysis of a range of over-the-counter and prescription tablet formations was demonstrated for amphetamine (methylphenidate), antidepressant (venlafaxine), barbiturate (Barbituric acid), depressant (alprazolam), narcotic (3-methylmorphine) and sympatholytic (propranolol) drugs. Active ingredients from soft and liquid formulations, such as Icy Hot cream (methyl salicylate) and Nyquil cold medicine (acetaminophen, dextromethorphan, doxylamine) were also detected. Increased sensitivity for selective drug responses was demonstrated through the formation of sodiated adduct ions by introducing small quantities of NaCl into the DESI solvent. Of the drugs and pharmaceuticals tested in this study, 68% (22 total samples) provided a clear ion mobility response at characteristic mobilities either as (M + H)(+), (M - H)(-), or (M + Na)(+) ions.

Carbohydrate structure characterization by tandem ion mobility mass spectrometry (IMMS)2.

A high resolution ion mobility spectrometer was interfaced to a Synapt G2 high definition mass spectrometer (HDMS) to produce IMMS-IMMS analysis. The hybrid instrument contained an electrospray ionization source, two ion gates, an ambient pressure linear ion mobility drift tube, a quadrupole mass filter, a traveling wave ion mobility spectrometer (TWIMS), and a time-of-flight mass spectrometer. The dual gate drift tube ion mobility spectrometer (DTIMS) could be used to acquire traditional IMS spectra but also could selectively transfer specific mobility selected precursor ions to the Synapt G2 HDMS for mass filtration (quadrupole). The mobility and mass selected ions could then be introduced into a collision cell for fragmentation followed by mobility separation of the fragment ions with the traveling wave ion mobility spectrometer. These mobility separated fragment ions are finally mass analyzed using a time-of-flight mass spectrometer. This results in an IMMS-IMMS analysis and provides a method to evaluate the isomeric heterogeneity of precursor ions by both DTIMS and TWIMS to acquire a mobility-selected and mass-filtered fragmentation pattern and to additionally obtain traveling wave ion mobility spectra of the corresponding product ions. This new IMMS(2) instrument enables the structural diversity of carbohydrates to be studied in greater detail. The physical separation of isomeric oligosaccharide mixtures was achieved by both DTIMS and TWIMS, with DTIMS demonstrating higher resolving power (70-80) than TWIMS (30-40). Mobility selected MS/MS spectra were obtained, and TWIMS evaluation of product ions showed that isomeric forms of fragment ions existed for identical m/z values.

Analysis of psychoactive cathinones and tryptamines by electrospray ionization atmospheric pressure ion mobility time-of-flight mass spectrometry.

The ability to use positive ion monitoring mode with an atmospheric pressure ion mobility time-of-flight mass spectrometer (APIM(tof)MS) to detect psychoactive cathinones and tryptamines from aqueous phase samples was evaluated. The study used a traditional electrospray ionization (ESI) source for sample introduction and ionization. A total of four cathinones (mephedrone, butylone, 4-Me-PPP, and 4-MEC) and five tryptamines (5-EtO-DPT, 5-EtO-DALT, 5-EtO-MIPT, 5-EtO-ALCHT, and 5-EtO-2MALET) were investigated, and we report on parent ions, collision induced dissociation (CID) fragment ions, reduced mobility (Ko), mass flight times, and detection limits obtained from a single instrument run for the psychoactive substances. Detection limits reported ranged from 3 to 11 µM concentration for the compounds studied. This detection limit range corresponded to 1-5 ng of material needed for improved detection on the instrument. This article demonstrates that it was possible to use a single instrument platform for the separation, detection, and identification of cathinones and tryptamines in less than 1 min. The application holds great promise for detecting and identifying a new class of drugs often referred to as "bath salts" or "legal highs" distributed over the Internet.

Ion mobility mass spectrometry analysis of isomeric disaccharide precursor, product and cluster ions.

RATIONALE: Carbohydrates are highly variable in structure owing to differences in their anomeric configurations, monomer stereochemistry, inter-residue linkage positions and general branching features. The separation of carbohydrate isomers poses a great challenge for current analytical techniques. METHODS: The isomeric heterogeneity of disaccharide ions and monosaccharide-glycolaldehyde product ions was evaluated using electrospray traveling wave ion mobility mass spectrometry (Synapt G2 high-definition mass spectrometer) in both positive and negative ion modes. RESULTS: The separation of isomeric disaccharide ions was observed but not fully achieved based on their mobility profiles. The mobilities of isomeric product ions, the monosaccharide-glycolaldehydes, derived from different disaccharide isomers were measured. Multiple mobility peaks were observed for both monosaccharide-glycolaldehyde cations and anions, indicating that there was more than one structural configuration in the gas phase as verified by NMR in solution. More importantly, the mobility patterns for isomeric monosaccharide-glycolaldehyde product ions were different, which enabled partial characterization of their respective disaccharide ions. Abundant disaccharide cluster ions were also observed. The results showed that a majority of isomeric cluster ions had different drift times and, moreover, more than one mobility peak was detected for a number of specific cluster ions. CONCLUSIONS: It is demonstrated that ion mobility mass spectrometry is an advantageous method to assess the isomeric heterogeneity of carbohydrate compounds. It is capable of differentiating different types of carbohydrate ions having identical m/z values as well as multiple structural configurations of single compounds.

Metabolomics of colorectal cancer: past and current analytical platforms.

Metabolomics is coming of age as an important area of investigation which may help reveal answers to questions left unanswered or only partially understood from proteomic or genomic approaches. Increased knowledge of the relationship of genes and proteins to smaller biomolecules (metabolites) will advance our ability to diagnose, treat, and perhaps prevent cancer and other diseases that have eluded scientists for generations. Colorectal tumors are the second leading cause of cancer mortality in the USA, and the incidence is rising. Many patients present late, after the onset of symptoms, when the tumor has spread from the primary site. Once metastases have occurred, the prognosis is significantly worse. Understanding alterations in metabolic profiles that occur with tumor onset and progression could lead to better diagnostic tests as well as uncover new approaches to treat or even prevent colorectal cancer (CRC). In this review, we explore the various analytical technologies that have been applied in CRC metabolomics research and summarize all metabolites measured in CRC and integrate them into metabolic pathways. Early studies with nuclear magnetic resonance and gas-chromatographic mass spectrometry suggest that tumor cells are characterized by aerobic glycolysis, increased purine metabolism for DNA synthesis, and protein synthesis. Liquid chromatography, capillary electrophoresis, and ion mobility, each coupled with mass spectrometry, promise to advance the field and provide new insight into metabolic pathways used by cancer cells. Studies with improved technology are needed to identify better biomarkers and targets for treatment or prevention of CRC.

Neuronal metabolomics by ion mobility mass spectrometry: cocaine effects on glucose and selected biogenic amine metabolites in the frontal cortex, striatum, and thalamus of the rat.

We report results of studies of global and targeted neuronal metabolomes by ambient pressure ion mobility mass spectrometry. The rat frontal cortex, striatum, and thalamus were sampled from control nontreated rats and those treated with acute cocaine or pargyline. Quantitative evaluations were made by standard additions or isotopic dilution. The mass detection limit was ~100 pmol varying with the analyte. Targeted metabolites of dopamine, serotonin, and glucose followed the rank order of distribution expected between the anatomical areas. Data was evaluated by principal component analysis on 764 common metabolites (identified by m/z and reduced mobility). Differences between anatomical areas and treatment groups were observed for 53 % of these metabolites using principal component analysis. Global and targeted metabolic differences were observed between the three anatomical areas with contralateral differences between some areas. Following drug treatments, global and targeted metabolomes were found to shift relative to controls and still maintained anatomical differences. Pargyline reduced 3,4-dihydroxyphenylacetic acid below detection limits, and 5-HIAA varied between anatomical regions. Notable findings were: (1) global metabolomes were different between anatomical areas and were altered by acute cocaine providing a broad but targeted window of discovery for metabolic changes produced by drugs of abuse; (2) quantitative analysis was demonstrated using isotope dilution and standard addition; (3) cocaine changed glucose and biogenic amine metabolism in the anatomical areas tested; and (4) the largest effect of cocaine was on the glycolysis metabolome in the thalamus confirming inferences from previous positron emission tomography studies using 2-deoxyglucose.

Evaluation of ion mobility-mass spectrometry for determining the isomeric heterogeneity of oligosaccharide-alditols derived from bovine submaxillary mucin.

Rapid separation and independent analysis of isomeric species are needed for the structural characterization of carbohydrates in glycomics research. Ion mobility-mass spectrometry techniques were used to examine a series of isomeric neutral oligosaccharide-alditols derived from bovine submaxillary mucin. Several analytical techniques were employed: (1) off line separation of the oligosaccharide-alditol mixture by HPLC; (2) direct and rapid evaluation of isomeric heterogeneity of oligosaccharides by electrospray ionization-ion mobility-time of flight mass spectrometry; and (3) mobility-selected MS2 and MS3 to evaluate isomeric mobility peaks by dual gate ion mobility-tandem mass spectrometry. Multiple isomeric ion mobility peaks were observed for the majority of oligosaccharide-alditols, which was achieved on the millisecond time scale after LC separation. Fragmentation spectra obtained from the collision-induced dissociation of isomeric precursor ions could be essentially identical, or dramatically different for a given precursor m/z using the dual-gate ion mobility quadrupole ion trap mass spectrometer. This further confirmed the need for rapid physical resolution of isomeric precursor species prior to their tandem mass spectral analysis.

Potential role of α-synuclein in neurodegeneration: studies in a rat animal model.

Neuronal protein α-synuclein (α-syn) is an essential player in the development of neurodegenerative diseases called synucleinopathies. A spontaneous autosomal recessive rat model for neurodegeneration was developed in our laboratory. These rats demonstrate progressive increases in α-syn in the brain mesencephalon followed by loss of dopaminergic terminals in the basal ganglia (BG) and motor impairments. The severity of pathology is directly related to the overexpression of α-syn and parallel decrease in dopamine (DA) level in the striatum (ST) of affected rats. The neurodegeneration in this model is characterized by the presence of perikarya and neurites Lewis bodies (LB) and diffuse marked accumulation of perikaryal α-syn in the substantia nigra (SN), brain stem (BS), and striatum (ST) along with neuronal loss. Light and ultrastructural analyses revealed that the process of neuronal degeneration is a 'dying back' type. The disease process is accompanied by gliosis and release of inflammatory cytokines. This neurodegeneration is a multisystemic disease and implicate α-syn as a major factor in the pathogenesis of this inherited autosomal recessive animal model. Decrease dopamine (DA) and overexpression of α-syn in the brain mesencephalon may provide a naturally occurring animal model for Parkinson's disease (PD) and other synucleinopathies that reproduces significant pathological, neurochemical, and behavioral features of the human disease.