An atypically mild case of ethylmalonic encephalopathy with pathogenic ETHE1 variant.

Ethylmalonic encephalopathy (EE) is a rare, severe, autosomal recessive condition caused by pathogenic variants in ETHE1 leading to progressive encephalopathy, hypotonia evolving to dystonia, petechiae, orthostatic acrocyanosis, diarrhea, and elevated ethylmalonic acid in urine. In this case report, we describe a patient with only mild speech and gross motor delays, subtle biochemical abnormalities, and normal brain imaging found to be homozygous for a pathogenic ETHE1 variant (c.586G>A) via whole exome sequencing. This case highlights the clinical heterogeneity of ETHE1 mutations and the utility of whole-exome sequencing in diagnosing mild cases of EE.

Synaptic Plasticity in the Nucleus Accumbens: Lessons Learned from Experience.

Synaptic plasticity contributes to behavioral adaptations. As a key node in the reward pathway, the nucleus accumbens (NAc) is important for determining motivation-to-action outcomes. Across animal models of motivation including addiction, depression, anxiety, and hedonic feeding, selective recruitment of neuromodulatory signals and plasticity mechanisms have been a focus of physiologists and behaviorists alike. Experience-dependent plasticity mechanisms within the NAc vary depending on the distinct afferents and cell-types over time. A greater understanding of molecular mechanisms determining how these changes in synaptic strength track with behavioral adaptations will provide insight into the process of learning and memory along with identifying maladaptations underlying pathological behavior. Here, we summarize recent findings detailing how changes in NAc synaptic strength and mechanisms of plasticity manifest in various models of motivational disorders.

Toll-like receptor 4 deficiency alters nucleus accumbens synaptic physiology and drug reward behavior.

Behavioral manifestations of drug-seeking behavior are causally linked to alterations of synaptic strength onto nucleus accumbens (NAc) medium spiny neurons (MSN). Although neuron-driven changes in physiology and behavior are well characterized, there is a lack of knowledge of the role of the immune system in mediating such effects. Toll-like receptor 4 (TLR4) is a pattern recognition molecule of the innate immune system, and evidence suggests that it modulates drug-related behavior. Using TLR4 knockout (TLR4.KO) mice, we show that TLR4 plays a role in NAc synaptic physiology and behavior. In addition to differences in the pharmacological profile of N-methyl-d-aspartate receptors (NMDAR) in the NAc core, TLR4.KO animals exhibit a deficit in low-frequency stimulation-induced NMDAR-dependent long-term depression (LTD). Interestingly, the synaptic difference is region specific as no differences were found in excitatory synaptic properties in the NAc shell. Consistent with altered NAc LTD, TLR4.KO animals exhibit an attenuation in drug reward learning. Finally, we show that TLR4 in the NAc core is primarily expressed on microglia. These results suggest that TLR4 influences NAc MSN synaptic physiology and drug reward learning and behavior.

Transgenic quail as a model for research in the avian nervous system: a comparative study of the auditory brainstem.

Research performed on transgenic animals has led to numerous advances in biological research. However, using traditional retroviral methods to generate transgenic avian research models has proved problematic. As a result, experiments aimed at genetic manipulations on birds have remained difficult for this popular research tool. Recently, lentiviral methods have allowed the production of transgenic birds, including a transgenic Japanese quail (Coturnix coturnix japonica) line showing neuronal specificity and stable expression of enhanced green fluorescent protein (eGFP) across generations (termed here GFP quail). To test whether the GFP quail may serve as a viable alternative to the popular chicken model system, with the additional benefit of genetic manipulation, we compared the development, organization, structure, and function of a specific neuronal circuit in chicken (Gallus gallus domesticus) with that of the GFP quail. This study focuses on a well-defined avian brain region, the principal nuclei of the sound localization circuit in the auditory brainstem, nucleus magnocellularis (NM), and nucleus laminaris (NL). Our results demonstrate that structural and functional properties of NM and NL neurons in the GFP quail, as well as their dynamic properties in response to changes in the environment, are nearly identical to those in chickens. These similarities demonstrate that the GFP quail, as well as other transgenic quail lines, can serve as an attractive avian model system, with the advantage of being able to build on the wealth of information already available from the chicken.

Pre-target axon sorting in the avian auditory brainstem.

Topographic organization of neurons is a hallmark of brain structure. The establishment of the connections between topographically organized brain regions has attracted much experimental attention, and it is widely accepted that molecular cues guide outgrowing axons to their targets in order to construct topographic maps. In a number of systems afferent axons are organized topographically along their trajectory as well, and it has been suggested that this pre-target sorting contributes to map formation. Neurons in auditory regions of the brain are arranged according to their best frequency (BF), the sound frequency they respond to optimally. This BF changes predictably with position along the so-called tonotopic axis. In the avian auditory brainstem, the tonotopic organization of the second- and third-order auditory neurons in nucleus magnocellularis (NM) and nucleus laminaris (NL) has been well described. In this study we examine whether the decussating NM axons forming the crossed dorsal cochlear tract (XDCT) and innervating the contralateral NL are arranged in a systematic manner. We electroporated dye into cells in different frequency regions of NM to anterogradely label their axons in XDCT. The placement of dye in NM was compared to the location of labeled axons in XDCT. Our results show that NM axons in XDCT are organized in a precise tonotopic manner along the rostrocaudal axis, spanning the entire rostrocaudal extent of both the origin and target nuclei. We propose that in the avian auditory brainstem, this pretarget axon sorting contributes to tonotopic map formation in NL.

Prolyl endopeptidase is revealed following SILAC analysis to be a novel mediator of human microglial and THP-1 cell neurotoxicity.

Reactive microglial cells may exacerbate the pathology in some neurodegenerative disorders. Supernatants of stimulated human microglial cells, or their surrogate THP-1 cells, are lethal to cultured human neuroblastoma SH-SY5Y cells. To explore this neurotoxicity, we examined the spectrum of proteins generated by THP-1 cells using the technique of stable isotope labeling by amino acids in cell culture (SILAC). Unstimulated cells were grown in medium with light L-[(12)C(6)] arginine while cells stimulated by lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma) were grown in medium with heavy L-[(13)C(6)] arginine. Proteins isolated from the media were digested with trypsin, and relative concentrations of generated peptides determined by mass spectrometry. More than 1,500 proteins or putative proteins were identified. Of these, 174 were increased and 189 decreased by more than twofold in the stimulated cell supernatant. We selected one upregulated protein, prolyl endopeptidase (PEP), for further investigation of its potential contribution to neurotoxicity. We first confirmed its upregulation by comparing its enzymatic activity in stimulated and unstimulated cell supernatants. We then evaluated two specific PEP inhibitors, Boc-Asn-Phe-Pro-aldehyde and Z-Pro-Pro-aldehyde-dimethyl acetal, for their potential to reduce toxicity of stimulated THP-1 cell and human microglia supernatants towards SH-SY5Y cells. We found both to be partially protective in a concentration-dependent manner. Inhibition of PEP may be a therapeutic approach to neurodegenerative disorders including Alzheimer and Parkinson diseases.

Identification of proteins in human substantia nigra.

Characterization of the human brain proteome is a critical area of research. While examination of the human cortex has provided some insight, very little is known about the proteome of the human midbrain, which demonstrates substantial loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) in Parkinson's disease (PD). Therefore, characterization of this region is essential to a better understanding of the pathogenesis of PD. This dataset paper reports two separate studies, where human SNpc was collected from PD and control subjects and 1263 proteins were identified using MALDI-TOF/TOF as well as linear ion trap MS platforms. With gene ontology analysis, the proteins were categorized according to their biological processes, as well as cellular components. These data were also compared with previous proteomic characterization of the human frontal and temporal cortex, and cerebrospinal fluid to establish shared proteins of relevance. The present dataset is the most extensive survey of the human SNpc proteome, to date. Further characterization of the SNpc proteome will significantly facilitate our understanding of the function and expression of proteins involved in PD, as well as provide potential proteins that may be utilized as biomarkers.

Identification of novel proteins affected by rotenone in mitochondria of dopaminergic cells.

BACKGROUND: Many studies have shown that mitochondrial dysfunction, complex I inhibition in particular, is involved in the pathogenesis of Parkinson's disease (PD). Rotenone, a specific inhibitor of mitochondrial complex I, has been shown to produce neurodegeneration in rats as well as in many cellular models that closely resemble PD. However, the mechanisms through which complex I dysfunction might produce neurotoxicity are as yet unknown. A comprehensive analysis of the mitochondrial protein expression profile affected by rotenone can provide important insight into the role of mitochondrial dysfunction in PD. RESULTS: Here, we present our findings using a recently developed proteomic technology called SILAC (stable isotope labeling by amino acids in cell culture) combined with polyacrylamide gel electrophoresis and liquid chromatography-tandem mass spectrometry to compare the mitochondrial protein profiles of MES cells (a dopaminergic cell line) exposed to rotenone versus control. We identified 1722 proteins, 950 of which are already designated as mitochondrial proteins based on database search. Among these 950 mitochondrial proteins, 110 displayed significant changes in relative abundance after rotenone treatment. Five of these selected proteins were further validated for their cellular location and/or treatment effect of rotenone. Among them, two were confirmed by confocal microscopy for mitochondrial localization and three were confirmed by Western blotting (WB) for their regulation by rotenone. CONCLUSION: Our findings represent the first report of these mitochondrial proteins affected by rotenone; further characterization of these proteins may shed more light on PD pathogenesis.

Proteomics identification of proteins in human cortex using multidimensional separations and MALDI tandem mass spectrometer.

It is essential to characterize the proteome of various regions of human brain because most, if not all, neurodegenerative diseases are region-specific. Here we report an in-depth proteomics identification of proteins extracted from the frontal cortex, a region playing a critical role in cognitive function. The integrated proteomics analytical flow consisted of biochemical fractionation, strong cation exchange chromatography, reverse phase liquid chromatography, and MALDI-TOF/TOF mass spectrometric analysis. In total, 812 proteins were confidently identified with two or more peptides. These proteins demonstrated diverse isoelectric points and molecular weights and are involved in several molecular functions, including protein binding, catalytic activity, transport, structure, and signal transduction. A number of proteins known to be associated with neurodegenerative diseases were also identified. Detailed characterization of these proteins will supply the necessary information to appropriately interpret proteins associated with aging and/or age-related neurodegenerative diseases. Finally 140 proteins found in the cortical proteome were present in the proteome of cerebrospinal fluid, providing tissue-specific candidates for biomarker discovery in body fluid.