Heterozygous loss of Engrailed-1 and α-synucleinopathy (En1/SYN): A dual-hit preclinical mouse model of Parkinson's disease, analyzed with artificial intelligence.

In this study, we develop and validate a new Parkinson's disease (PD) mouse model that can be used to better understand how the disease progresses and to test the effects of new, potentially disease-modifying, PD therapies. Our central hypothesis is that mitochondrial dysfunction intercalates with misfolded α-synuclein (α-syn) accumulation in a vicious cycle, leading to the loss of nigral neurons. Our hypothesis builds on the concept that PD involves multiple molecular insults, including mitochondrial dysfunction and aberrant α-syn handling. We predicted that mitochondrial deficits, due to heterozygous loss of Engrailed-1 (En1+/-), combined with bilateral injections of pathogenic α-syn fibrils (PFFs), will act to generate a highly relevant PD model - the En1/SYN model. Here, En1+/- mice received bilateral intrastriatal stereotaxic injections of either PBS or α-syn fibrils and were analyzed using automated behavioral tests and deep learning-assisted histological analysis at 2, 4, and 6 months post-injection. We observed significant and progressive Lewy body-like inclusion pathology in the amygdala, motor cortex, and cingulate cortex, as well as the loss of tyrosine hydroxylase-positive (TH+) cells in the substantia nigra. The En1/SYN model also exhibited significant motor impairments at 6 months post-injection, which were however not exacerbated as we had expected. Still, this model has a comprehensive number of PD-like phenotypes and is therefore superior when compared to the α-syn PFF or En1+/- models alone.

Urinary tract infections trigger synucleinopathy via the innate immune response.

Symptoms in the urogenital organs are common in multiple system atrophy (MSA), also in the years preceding the MSA diagnosis. It is unknown how MSA is triggered and these observations in prodromal MSA led us to hypothesize that synucleinopathy could be triggered by infection of the genitourinary tract causing ɑ-synuclein (ɑSyn) to aggregate in peripheral nerves innervating these organs. As a first proof that peripheral infections could act as a trigger in MSA, this study focused on lower urinary tract infections (UTIs), given the relevance and high frequency of UTIs in prodromal MSA, although other types of infection might also be important triggers of MSA. We performed an epidemiological nested-case control study in the Danish population showing that UTIs are associated with future diagnosis of MSA several years after infection and that it impacts risk in both men and women. Bacterial infection of the urinary bladder triggers synucleinopathy in mice and we propose a novel role of ɑSyn in the innate immune system response to bacteria. Urinary tract infection with uropathogenic E. coli results in the de novo aggregation of ɑSyn during neutrophil infiltration. During the infection, ɑSyn is released extracellularly from neutrophils as part of their extracellular traps. Injection of MSA aggregates into the urinary bladder leads to motor deficits and propagation of ɑSyn pathology to the central nervous system in mice overexpressing oligodendroglial ɑSyn. Repeated UTIs lead to progressive development of synucleinopathy with oligodendroglial involvement in vivo. Our results link bacterial infections with synucleinopathy and show that a host response to environmental triggers can result in ɑSyn pathology that bears semblance to MSA.

Low prevalence of ideal levels in cardiovascular behavior metrics among Mexican adolescents.

BACKGROUND: Lifestyle changes when transitioning from high-school to college expose students to unhealthy behaviors associated with high cardiovascular risk. The study aimed to assess the cardiovascular behavior metrics according to the AHA criteria, in freshman college adolescents from Northwest Mexico. METHODS: The study was cross-sectional. Demographics and health history were collected by questionnaires. Four behaviors were evaluated: diet quality using a duplicated FFQ, physical activity (PA) using the IPAQ, smoking, and body mass index (BMI) percentile; blood pressure was measured as a biological metric. Intakes were averaged and summed for each food group; sodium and saturated fat were calculated using the Mexican System of Food Equivalents or the USDA Database. Metrics were categorized into ideal, intermediate or poor level according to the AHA criteria. Diet outliers (± 3 SD) were trimmed and data was tested for normality. Mean±SD were calculated for continuous and percentages for categorical variables. Chi-square test compared the prevalence of demographic variables and levels of each cardiovascular metric by sex. Independent T-test evaluated differences in anthropometrics, dietary, and PA by sex, and the prevalence of ideal vs. non-ideal dietary intakes. RESULTS: Participants were n = 228, 55.6% men, age = 18.5±0.4 y. A higher prevalence of men indicated working, playing sports, and family history hypertriglyceridemia (p < 0.05). Men showed higher weight, height, BMI, waist, blood pressure, and lower PA and body fat (p < 0.05). Concerning diet quality, significant differences by sex were observed in nuts and seeds (1.1±0.6 and 0.9±0.6 oz/week, p = 0.042) and processed meats (749.8±639 and 503.6±300.3 g/week, p = 0.002); only fish and shellfish group reached AHA recommendations (513.1 ± 450.7 vs. 501.7 ± 428 g/week, p = 0.671) for men and women, respectively. Ideal level was reached by 70.9% participants for BMI percentile, 87% for smoking, 67.2% for blood pressure, 25.9% for PA, and 12.2% for diet score. Regarding food groups and nutrients, the lower prevalence in the ideal level was for sugar-sweetened beverages (10%, p = 0.013) and processed meats (4.8%, p = 0.208), and the highest for fish and shellfish (87.8%, p = 0.281) . CONCLUSIONS: The diet and PA patterns of Northwest Mexican freshman adolescents make them a high-risk group for developing long-term unhealthy habits and cardiovascular complications early in adulthood.

ERCC4 variants identified in a cohort of patients with segmental progeroid syndromes.

Pathogenic variants in genes, which encode DNA repair and damage response proteins, result in a number of genomic instability syndromes with features of accelerated aging. ERCC4 (XPF) encodes a protein that forms a complex with ERCC1 and is required for the 5' incision during nucleotide excision repair. ERCC4 is also FANCQ, illustrating a critical role in interstrand crosslink repair. Pathogenic variants in this gene cause xeroderma pigmentosum, XFE progeroid syndrome, Cockayne syndrome (CS), and Fanconi anemia. We performed massive parallel sequencing for 42 unsolved cases submitted to the International Registry of Werner Syndrome. Two cases, each carrying two novel heterozygous ERCC4 variants, were identified. The first case was a compound heterozygote for: c.2395C > T (p.Arg799Trp) and c.388+1164_792+795del (p.Gly130Aspfs*18). Further molecular and cellular studies indicated that the ERCC4 variants in this patient are responsible for a phenotype consistent with a variant of CS. The second case was heterozygous for two variants in cis: c.[1488A > T; c.2579C > A] (p.[Gln496His; Ala860Asp]). While the second case also had several phenotypic features of accelerated aging, we were unable to provide biological evidence supporting the pathogenic roles of the associated ERCC4 variants. Precise genetic causes and disease mechanism of the second case remains to be determined.

Targeting PERK signaling with the small molecule GSK2606414 prevents neurodegeneration in a model of Parkinson's disease.

Parkinson's disease (PD) is the second most common neurodegenerative disorder, leading to the progressive decline of motor control due to the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). Accumulating evidence suggest that altered proteostasis is a salient feature of PD, highlighting perturbations to the endoplasmic reticulum (ER), the main compartment involved in protein folding and secretion. PERK is a central ER stress sensor that enforces adaptive programs to recover homeostasis through a block of protein translation and the induction of the transcription factor ATF4. In addition, chronic PERK signaling results in apoptosis induction and neuronal dysfunction due to the repression in the translation of synaptic proteins. Here we confirmed the activation of PERK signaling in postmortem brain tissue derived from PD patients and three different rodent models of the disease. Pharmacological targeting of PERK by the oral administration of GSK2606414 demonstrated efficient inhibition of the pathway in the SNpc after experimental ER stress stimulation. GSK2606414 protected nigral-dopaminergic neurons against a PD-inducing neurotoxin, improving motor performance. The neuroprotective effects of PERK inhibition were accompanied by an increase in dopamine levels and the expression of synaptic proteins. However, GSK2606414 treated animals developed secondary effects possibly related to pancreatic toxicity. This study suggests that strategies to attenuate ER stress levels may be effective to reduce neurodegeneration in PD.

A new model to study cell-to-cell transfer of αSynuclein in vivo.

Parkinson's disease (PD) compromises motor control due to the loss of dopaminergic neurons in the substantia nigra pars compacta. At the histopathological level, PD is characterized by the accumulation of Lewy bodies, large protein inclusions containing aggregated αSynuclein (αSyn). The progression of PD involves the spreading of αSyn misfolding through the brain mediated by a prion-like mechanism, where the protein is transferred between cells. Here we report that αSyn internalization is a dynamic process, where the protein transits through different sub-cellular compartments. Importantly, cells incorporating αSyn develop larger protein-like inclusions when compared to αSyn producing cells. We developed a new tool to monitor cell-to-cell transfer of αSyn in vivo using an adeno-associated viral (AAV) vector expressing αSyn fused to a red fluorescent protein in addition to soluble EGFP to label donor cells. Intra-nigral delivery of this reporter AAV construct allowed the visualization of αSyn incorporation into surrounding neurons. This work provides a new tool to study αSyn cell-to-cell transfer in vivo and may open new opportunities to study PD pathogenesis.

Control of dopaminergic neuron survival by the unfolded protein response transcription factor XBP1.

Parkinson disease (PD) is characterized by the selective loss of dopaminergic neurons of the substantia nigra pars compacta (SNpc). Although growing evidence indicates that endoplasmic reticulum (ER) stress is a hallmark of PD, its exact contribution to the disease process is not well understood. Here we report that developmental ablation of X-Box binding protein 1 (XBP1) in the nervous system, a key regulator of the unfolded protein response (UPR), protects dopaminergic neurons against a PD-inducing neurotoxin. This survival effect was associated with a preconditioning condition that resulted from induction of an adaptive ER stress response in dopaminergic neurons of the SNpc, but not in other brain regions. In contrast, silencing XBP1 in adult animals triggered chronic ER stress and dopaminergic neuron degeneration. Supporting this finding, gene therapy to deliver an active form of XBP1 provided neuroprotection and reduced striatal denervation in animals injected with 6-hydroxydopamine. Our results reveal a physiological role of the UPR in the maintenance of protein homeostasis in dopaminergic neurons that may help explain the differential neuronal vulnerability observed in PD.

Microcin e492 amyloid formation is retarded by posttranslational modification.

Microcin E492, a channel-forming bacteriocin with the ability to form amyloid fibers, is exported as a mixture of two forms: unmodified (inactive) and posttranslationally modified at the C terminus with a salmochelin-like molecule, which is an essential modification for conferring antibacterial activity. During the stationary phase, the unmodified form accumulates because expression of the maturation genes mceIJ is turned off, and microcin E492 is rapidly inactivated. The aim of this work was to demonstrate that the increase in the proportion of unmodified microcin E492 augments the ability of this bacteriocin to form amyloid fibers, which in turn decreases antibacterial activity. To this end, strains with altered proportions of the two forms were constructed. The increase in the expression of the maturation genes augmented the antibacterial activity during all growth phases and delayed the loss of activity in the stationary phase, while the ability to form amyloid fibers was markedly reduced. Conversely, a higher expression of microcin E492 protein produced concomitant decreases in the levels of the modified form and in antibacterial activity and a substantial increase in the ability to form amyloid fibers. The same morphology for these fibers, including those formed by only the unmodified version, was observed. Moreover, seeds formed using exclusively the nonmodified form were remarkably more efficient in amyloid formation with a shorter lag phase, indicating that the nucleation process is probably improved. Unmodified microcin E492 incorporation into amyloid fibers was kinetically more efficient than the modified form, probably due to the existence of a conformation that favors this process.

Distribution of phosphorylated alpha-synuclein in non-diseased brain implicates olfactory bulb mitral cells in synucleinopathy pathogenesis.

Synucleinopathies are neurodegenerative diseases characterized by pathological inclusions called "Lewy pathology" (LP) that consist of aggregated alpha-synuclein predominantly phosphorylated at serine 129 (PSER129). Despite the importance for understanding disease, little is known about the endogenous function of PSER129 or why it accumulates in the diseased brain. Here we conducted several observational studies using a sensitive tyramide signal amplification (TSA) technique to determine PSER129 distribution and function in the non-diseased mammalian brain. In wild-type non-diseased mice, PSER129 was detected in the olfactory bulb (OB) and several brain regions across the neuroaxis (i.e., OB to brainstem). In contrast, PSER129 immunoreactivity was not observed in any brain region of alpha-synuclein knockout mice. We found evidence of PSER129 positive structures in OB mitral cells of non-diseased mice, rats, non-human primates, and healthy humans. Using TSA multiplex fluorescent labeling, we showed that PSER129 positive punctate structures occur within inactive (i.e., c-fos negative) T-box transcription factor 21 (TBX21) positive mitral cells and PSER129 within these cells was spatially associated with PK-resistant alpha-synuclein. Ubiquitin was found in PSER129 mitral cells but was not closely associated with PSER129. Biotinylation by antibody recognition (BAR) identified 125 PSER129-interacting proteins in the OB of healthy mice, which were significantly enriched for presynaptic vesicle trafficking/recycling, SNARE, fatty acid oxidation, oxidative phosphorylation, and RNA binding. TSA multiplex labeling confirmed the physical association of BAR-identified protein Ywhag with PSER129 in the OB and in other regions across the neuroaxis. We conclude that PSER129 accumulates in the mitral cells of the healthy OB as part of alpha-synuclein normal cellular functions. Incidental LP has been reported in the OB, and therefore we speculate that for synucleinopathies, either the disease processes begin locally in OB mitral cells or a systemic disease process is most apparent in the OB because of the natural tendency to accumulate PSER129.

Associations between dietary intake and physical activity, as behavioral indicators, with body fat in adolescents from Mexico.

Introduction: Introduction: the prevention of cardiometabolic diseases is currently a priority due to its relationship with COVID-19 complications. Unhealthy lifestyles, like inadequate diet and physical inactivity, are the cornerstone for obesity and cardiometabolic risk. Objective: to examine the association between diet and physical activity with body fat in Mexican adolescents. Methods: the study was cross-sectional. Data included socio-demographic variables, health history and smoking habit obtained through questionnaires; blood pressure and anthropometry measurements; food and nutrient intake through 24-hour recalls; and physical activities through the International Physical Activity Questionnaire (IPAQ). Univariate analyses were used and multiple models were built by stepwise forward selection (p ≤ 0.05 and biological plausibility). Results: participants were n = 230 students, 18.5 ± 0.4 years and 54.8 % men. Three models associated with body fat were obtained. In the first model, saturated fatty acids (ß = 0.30, p = 0.028) were significant positive associated, while vigorous physical activity was significant negative associated as a protective factor (ß = -0.007, p = 0.023). In the second model, total fat (ß = 0.17, p = 0.005) was significant positive associated, and vigorous physical activity was significant negative associated (ß = -0.007, p = 0.023). The third model included fruits and vegetables (ß = -5.49, p = 0.092) and vigorous physical activity (ß = -0.006, p = 0.058) as protective factors. Conclusions: dietary intake of total fat and saturated fatty acids, and vigorous physical activity, were significantly associated with body fat, while fruits and vegetables trended toward significance, in Mexican adolescents. Community-based programs that promote intake of protective foods and reduction of risky foods, and encourage vigorous physical activity, are needed in adolescents.

Introducción: Introducción: la prevención de enfermedades cardiometabólicas es actualmente una prioridad por su relación con las complicaciones de la COVID-19. Los estilos de vida poco saludables son la piedra angular de la obesidad y el riesgo cardiometabólico. Objetivo: examinar la asociación entre dieta y actividad física con la grasa corporal en adolescentes mexicanos. Métodos: el estudio fue transversal. Los datos incluyeron variables sociodemográficas; mediciones de presión arterial y antropometría; ingesta de alimentos y nutrimentos con recordatorios de 24 horas; y actividades físicas con el cuestionario internacional de actividad física (IPAQ). Se utilizo análisis univariado y los modelos múltiples se construyeron mediante selección paso a paso hacia adelante (p ≤ 0.05 y plausibilidad biológica). Resultados: participaron n = 230 estudiantes, 18,5 ± 0,4 años y 54,8 % hombres. Se obtuvieron tres modelos asociados a la grasa corporal. En el primero, los ácidos grasos saturados (ß = 0,30, p = 0,028) se asociaron significativamente de forma positiva, y la actividad física vigorosa significativamente de forma negativa como factor protector (ß = -0,007, p = 0,023). En el segundo, la grasa total (ß = 0,17, p = 0,005) se asoció positivamente y la actividad física vigorosa, negativamente (ß = -0,007, p = 0,023). El tercer modelo incluyó frutas y verduras (ß = -5,49, p = 0,092) y actividad física vigorosa (ß = -0,006, p = 0,058) como factores protectores. Conclusiones: la ingesta dietética de grasas totales y saturadas y la actividad física vigorosa se asociaron significativamente con la grasa corporal, mientras que las frutas y verduras tendieron a ser significativas. Se necesitan programas comunitarios que promuevan la ingesta de alimentos protectores y la reducción de alimentos de riesgo, y fomenten la actividad física vigorosa en adolescentes.

Structural characterization of microcin E492 amyloid formation: Identification of the precursors.

Microcin E492 is a low-molecular weight, channel-forming bacteriotoxin that generates amyloid structures. Using electron microscopy and image processing techniques several structural conformations can be observed. Prior to the conditions that induce amyloid formation and at its initial stage, microcin E492 molecules can be found in two main types of oligomers: a pentameric, pore-like structure consisting of globular monomers of ∼25Å diameter, and long filaments made up of stacked pentamers. The equilibrium between these structures depends on the properties of the solvent, because samples kept in methanol mainly show the pentameric structure. Amyloid induction in aqueous solvent reveals the presence, together with the above mentioned structures, of several amyloid structures such as flat and helical filaments. In addition, X-ray diffraction analysis demonstrated that the fibrils formed by microcin E492 presented cross-ß structure, a distinctive property of amyloid fibrils. Based on the study of the observed structures we propose that microcin E492 has two conformations: a native one that assembles mainly into a pentameric structure, which functions as a pore, and an amyloid conformation which results in the formation of different types of amyloid filaments.

Cementum protein 1 (CEMP1) induces differentiation by human periodontal ligament cells under three-dimensional culture conditions.

PDL (periodontal ligament) is a source of multi-potent stem cells in humans and their differentiation potential to a cementoblast and osteoblast phenotypes has been shown. Tissue construction from PDL-derived cells could be considered as a valuable technique for periodontal regenerative medicine. On these basis, we determined the role of CEMP1 (cementum protein 1) as a factor to induce differentiation of human PDL cells in a 3D (three-dimensional) fashion. Human PDL cells were grown in an RCCS (rotary cell culture system) D-410 RWV (rotating wall vessel) bioreactor, and maintained in either experimental (CEMP1 2.5 µg/ml) or control media during 4 weeks. Cell proliferation in the presence of CEMP1 was determined. The tissue-like structure formed was analysed histologically, stained with Alizarin Red and Alcian Blue. ALP (alkaline phosphatase)-specific activity, immunostaining, RT-PCR (reverse transcription-PCR) and Western blotting were performed to determine the expression of BSP (bone sialoprotein), enamel [AMBN (ameloblastin) and AMEL (amelogenin)], cementum [CAP (cementum attachment protein) and CEMP1] and cartilage-related proteins (Sox9, aggrecan, types II and X collagens). Our results show that hrCEMP1 (human recombinant CEMP1) promoted cell proliferation by human PDL cells in 3D cultures and induced the formation of a tissue-like structure resembling bone and/or cementum and material similar to cartilage. The addition of hrCEMP1 to the 3D human PDL cell cultures increased ALP-specific activity by 2.0-fold and induced the expression of markers for the osteogenic, cementogenic and chondrogenic phenotypes at the mRNA and protein levels. Our data show that human PDL cells in 3D cultures with the addition of CEMP1 has the potential to be used for the bioengineering reconstruction of periodontal tissues and cartilage since our results suggest that CEMP1 stimulates human PDL cells to differentiate towards different phenotypes.

Motivations and Barriers to Participation in a Randomized Trial on Melanoma Genomic Risk: A Mixed-Methods Analysis.

The evolution of polygenic scores for use in for disease prevention and control compels the development of guidelines to optimize their effectiveness and promote equitable use. Understanding the motivations and barriers to participation in genomics research can assist in drafting these standards. We investigated these in a community-based randomized controlled trial that examined the health behavioral impact of receiving personalized melanoma genomic risk information. We examined participant responses in a baseline questionnaire and conducted interviews post-trial participation. Motivations differed in two ways: (1) by gender, with those identifying as women placing greater importance on learning about their personal risk or familial risk, and how to reduce risk; and (2) by age in relation to learning about personal risk, and fear of developing melanoma. A barrier to participation was distrust in the handling of genomic data. Our findings provide new insights into the motivations for participating in genomics research and highlight the need to better target population subgroups including younger men, which will aid in tailoring recruitment for future genomic studies.

A novel automated morphological analysis of Iba1+ microglia using a deep learning assisted model.

There is growing evidence for the key role of microglial functional state in brain pathophysiology. Consequently, there is a need for efficient automated methods to measure the morphological changes distinctive of microglia functional states in research settings. Currently, many commonly used automated methods can be subject to sample representation bias, time consuming imaging, specific hardware requirements and difficulty in maintaining an accurate comparison across research environments. To overcome these issues, we use commercially available deep learning tools Aiforia® Cloud (Aifoira Inc., Cambridge, MA, United States) to quantify microglial morphology and cell counts from histopathological slides of Iba1 stained tissue sections. We provide evidence for the effective application of this method across a range of independently collected datasets in mouse models of viral infection and Parkinson's disease. Additionally, we provide a comprehensive workflow with training details and annotation strategies by feature layer that can be used as a guide to generate new models. In addition, all models described in this work are available within the Aiforia® platform for study-specific adaptation and validation.

Reproducibility and validity of a food frequency questionnaire to assess cardiovascular health-related food intake among Mexican adolescents.

There is a lack of region-adapted tools to evaluate diet as a risk factor for cardiovascular disease (CVD) in adolescents. The study aim was to evaluate the reproducibility and validity of a paper-based and region-adapted food frequency questionnaire (FFQ) designed to assess CVD-related food and nutrient intakes of adolescents from Northwest México. The study design was cross-sectional. The FFQ was developed in a two-step process: prototype designing and a pilot test, with re-tested in a 3-month period, along with two administrations of 24 h-recall (24 hR). Pearson's and intra-class correlation coefficients (PCC and ICC) were assessed. Bland-Altman plots, limits of agreement and quintile classifications were carried out. Participants (n 221) were 53·8 % male, 18·5 ± 0·4 years old. Reproducibility had a median PCC = 0·66 for processed meats, ranging from 0·40 (saturated fat) to 0·74 (fish & shellfish), P = 0·001. ICC ranged from 0·53 (saturated fat) to 0·80 (sodium; and nuts, seeds and legumes), P = 0·001. Validity comparing FFQ1 v. 24 hR mean, PCCs ranged from 0·12 (P = 0·06) to 0·95 (P = 0·001), and ICC from 0·20 (P = 0·048) to 0·88 (P = 0·001); comparing FFQ2 v. 24 hR mean, PCCs ranged from 0·07 (P = 0·25) to 0·46 (P = 0·001), and ICC from 0·15 (P = 0·106) to 0·58 (P = 0·001). The FFQ overestimated the intake of all food groups and nutrients (P < 0·05), while Cohen's κ showed coefficients lower than 0·20. The proposed FFQ represents a moderately validated tool to estimate CVD-related food and nutrient intakes as a risk factor, which can be used in combination with multiple administrations of 24 hRs, as a critical mean in future interventions intended to reduce cardiometabolic risk in adolescents.

Neuronal VPS35 deletion induces spinal cord motor neuron degeneration and early post-natal lethality.

Neurodegenerative diseases are characterized by the selective degeneration of neuronal populations in different brain regions and frequently the formation of distinct protein aggregates that often overlap between diseases. While the causes of many sporadic neurodegenerative diseases are unclear, genes associated with familial or sporadic forms of disease and the underlying cellular pathways involved tend to support common disease mechanisms. Underscoring this concept, mutations in the Vacuolar Protein Sorting 35 Orthologue (VPS35) gene have been identified to cause late-onset, autosomal dominant familial Parkinson's disease, whereas reduced VPS35 protein levels are reported in vulnerable brain regions of subjects with Alzheimer's disease, neurodegenerative tauopathies such as progressive supranuclear palsy and Pick's disease, and amyotrophic lateral sclerosis. Therefore, VPS35 is commonly implicated in many neurodegenerative diseases. VPS35 plays a critical role in the retromer complex that mediates the retrieval and recycling of transmembrane protein cargo from endosomes to the trans-Golgi network or plasma membrane. VPS35 and retromer function are highly conserved in eukaryotic cells, with the homozygous deletion of VPS35 inducing early embryonic lethality in mice that has hindered an understanding of its role in the brain. Here, we develop conditional knockout mice with the selective deletion of VPS35 in neurons to better elucidate its role in neuronal viability and its connection to neurodegenerative diseases. Surprisingly, the pan-neuronal deletion of VPS35 induces a progressive and rapid disease with motor deficits and early post-natal lethality. Underlying this neurological phenotype is the relatively selective and robust degeneration of motor neurons in the spinal cord. Neuronal loss is accompanied and preceded by the formation of p62-positive protein inclusions and robust reactive astrogliosis. Our study reveals a critical yet unappreciated role for VPS35 function in the normal maintenance and survival of motor neurons during post-natal development that has important implications for neurodegenerative diseases, particularly amyotrophic lateral sclerosis.

An extended release GLP-1 analogue increases α-synuclein accumulation in a mouse model of prodromal Parkinson's disease.

The repurposing of drugs developed to treat type 2 diabetes for the treatment of Parkinson's disease (PD) was encouraged by the beneficial effect exerted by the glucagon-like peptide 1 (GLP-1) analogue exenatide in a phase 2 clinical trial. The effects of GLP-1 analogues have been investigated extensively using rodent toxin models of PD. However, many of the toxin-based models used lack robust α-synuclein (α-syn) pathology, akin to the Lewy bodies and neurites seen in PD. One prior study has reported a protective effect of a GLP-1 analogue on midbrain dopamine neurons following injection of α-syn preformed fibrils (PFF) into the striatum. Here, we used olfactory bulb injections of PFF as a model of prodromal PD and monitored the effect of a long-acting GLP-1 analogue on the propagation of α-syn pathology in the olfactory system. Thirteen weeks after PFF injection, mice treated with long-acting the GLP-1 analogue had a significant increase in pathological α-syn in brain regions connected to the olfactory bulb, accompanied by signs of microglia activation. Our results suggest that the nature of the neuronal insult and intrinsic properties of the targeted neuronal population markedly influence the effect of GLP-1 analogues.

Deficits in olfactory sensitivity in a mouse model of Parkinson's disease revealed by plethysmography of odor-evoked sniffing.

Hyposmia is evident in over 90% of Parkinson's disease (PD) patients. A characteristic of PD is intraneuronal deposits composed in part of α-synuclein fibrils. Based on the analysis of post-mortem PD patients, Braak and colleagues suggested that early in the disease α-synuclein pathology is present in the dorsal motor nucleus of the vagus, as well as the olfactory bulb and anterior olfactory nucleus, and then later affects other interconnected brain regions. Here, we bilaterally injected α-synuclein preformed fibrils into the olfactory bulbs of wild type male and female mice. Six months after injection, the anterior olfactory nucleus and piriform cortex displayed a high α-synuclein pathology load. We evaluated olfactory perceptual function by monitoring odor-evoked sniffing behavior in a plethysmograph at one-, three- and six-months after injection. No overt impairments in the ability to engage in sniffing were evident in any group, suggesting preservation of the ability to coordinate respiration. At all-time points, females injected with fibrils exhibited reduced odor detection sensitivity, which was observed with the semi-automated plethysmography apparatus, but not a buried pellet test. In future studies, this sensitive methodology for assessing olfactory detection deficits could be used to define how α-synuclein pathology affects other aspects of olfactory perception and to clarify the neuropathological underpinnings of these deficits.

Simultaneous extraction of antibiotic and estrogen from animal blood serum using aqueous two-phase systems as predictor of environmental impact.

Efficient analytical methods are required for optimizing dosage of veterinary antibiotics and hormones in order to reduce toxicity and antimicrobial resistance in the environment. Thus, the objective of this work was to develop a rapid and low-cost method for determination of hormone estradiol and antibiotic chlortetracycline in bovine and porcine blood serum by aqueous two-phase system (ATPS) extraction and capillary electrophoresis quantification. ATPS based on ionic liquid cholinium alaninate and citrate salt along with mixtures of protic and aprotic polar solvents were evaluated in terms of recovery of extraction (%R). The liquid-liquid equilibrium, phase diagrams, and tie lines are discussed. Antibiotic migrated to solvent-rich phase (R ≈ 89.0%) to all systems. Estradiol migrates to ionic liquid-rich phase; however, addition of 10% methanol changed partition to solvent-rich phase (R ≈ 89.7%). The method has high recovery and cleanliness, is cost-efficient, scalable, and hence is adequate for screening of antibiotics and hormones tested in animal blood serum for dosage optimization and to predict their environment.