Mechanistic divergences of endocytic clathrin-coated vesicle formation in mammals, yeasts and plants.

Clathrin-coated vesicles (CCVs), generated by clathrin-mediated endocytosis (CME), are essential eukaryotic trafficking organelles that transport extracellular and plasma membrane-bound materials into the cell. In this Review, we explore mechanisms of CME in mammals, yeasts and plants, and highlight recent advances in the characterization of endocytosis in plants. Plants separated from mammals and yeast over 1.5 billion years ago, and plant cells have distinct biophysical parameters that can influence CME, such as extreme turgor pressure. Plants can therefore provide a wider perspective on fundamental processes in eukaryotic cells. We compare key mechanisms that drive CCV formation and explore what these mechanisms might reveal about the core principles of endocytosis across the tree of life. Fascinatingly, CME in plants appears to more closely resemble that in mammalian cells than that in yeasts, despite plants being evolutionarily further from mammals than yeast. Endocytic initiation appears to be highly conserved across these three systems, requiring similar protein domains and regulatory processes. Clathrin coat proteins and their honeycomb lattice structures are also highly conserved. However, major differences are found in membrane-bending mechanisms. Unlike in mammals or yeast, plant endocytosis occurs independently of actin, highlighting that mechanistic assumptions about CME across different systems should be made with caution.

Melatonin and Seasonal Synchrony in Mammals.

In mammals, seasonal opportunities and challenges are anticipated through programmed changes in physiology and behavior. Appropriate anticipatory timing depends on synchronization to the external solar year, achieved through the use of day length (photoperiod) as a synchronizing signal. In mammals, nocturnal production of melatonin by the pineal gland is the key hormonal mediator of photoperiodic change, exerting its effects via the hypothalamopituitary axis. In this review/perspective, we consider the key developments during the history of research into the seasonal synchronizer effect of melatonin, highlighting the role that the pars tuberalis-tanycyte module plays in this process. We go on to consider downstream pathways, which include discrete hypothalamic neuronal populations. Neurons that express the neuropeptides kisspeptin and (Arg)(Phe)-related peptide-3 (RFRP-3) govern seasonal reproductive function while neurons that express somatostatin may be involved in seasonal metabolic adaptations. Finally, we identify several outstanding questions, which need to be addressed to provide a much thorough understanding of the deep impact of melatonin upon seasonal synchronization.

Alzheimer's Disease Neuropathological Change in Aged Non-Primate Mammals.

Human brain aging is characterized by the production and deposition of ß-amyloid (Aß) in the form of senile plaques and cerebral amyloid angiopathy and the intracellular accumulation of hyper-phosphorylated tau (Hp-tau) to form neurofibrillary tangles (NFTs) and dystrophic neurites of senile plaques. The process progresses for years and eventually manifests as cognitive impairment and dementia in a subgroup of aged individuals. Aß is produced and deposited first in the neocortex in most aged mammals, including humans; it is usually not accompanied by altered behavior and cognitive impairment. Hp-tau is less frequent than Aß pathology, and NFTs are rare in most mammals. In contrast, NFTs are familiar from middle age onward in humans; NFTs first appear in the paleocortex and selected brain stem nuclei. NFTs precede for decades or years Aß deposition and correlate with dementia in about 5% of individuals at the age of 65 and 25% at the age of 85. Based on these comparative data, (a) Aß deposition is the most common Alzheimer's disease neuropathological change (ADNC) in the brain of aged mammals; (b) Hp-tau is less common, and NFTs are rare in most aged mammals; however, NFTs are the principal cytoskeletal pathology in aged humans; (c) NFT in aged humans starts in selected nuclei of the brain stem and paleocortical brain regions progressing to the most parts of the neocortex and other regions of the telencephalon; (d) human brain aging is unique among mammalian species due to the early appearance and dramatic progression of NFTs from middle age onward, matching with cognitive impairment and dementia in advanced cases; (e) neither mammalian nor human brain aging supports the concept of the amyloid cascade hypothesis.

Intercorrelations of Chlorinated Paraffins, Dechloranes, and Legacy Persistent Organic Pollutants in 10 Species of Marine Mammals from Norway, in Light of Dietary Niche.

Short-, medium-, and long-chain chlorinated paraffins (CPs) (SCCPs, MCCPs, and LCCPs) and dechloranes are chemicals of emerging concern; however, little is known of their bioaccumulative potential compared to legacy contaminants in marine mammals. Here, we analyzed SCCPs, MCCPs, LCCPs, 7 dechloranes, 4 emerging brominated flame retardants, and 64 legacy contaminants, including polychlorinated biphenyls (PCBs), in the blubber of 46 individual marine mammals, representing 10 species, from Norway. Dietary niche was modeled based on stable isotopes of nitrogen and carbon in the skin/muscle to assess the contaminant accumulation in relation to diet. SCCPs and dechlorane-602 were strongly positively correlated with legacy contaminants and highest in killer (Orcinus orca) and sperm (Physeter macrocephalus) whales (median SCCPs: 160 ng/g lw; 230 ng/g lw and median dechlorane-602: 3.8 ng/g lw; 2.0 ng/g lw, respectively). In contrast, MCCPs and LCCPs were only weakly correlated to recalcitrant legacy contaminants and were highest in common minke whales (Balaenoptera acutorostrata; median MCCPs: 480 ng/g lw and LCCPs: 240 ng/g lw). The total contaminant load in all species was dominated by PCBs and legacy chlorinated pesticides (63-98%), and MCCPs dominated the total CP load (42-68%, except 11% in the long-finned pilot whale Globicephala melas). Surprisingly, we found no relation between contaminant concentrations and dietary niche, suggesting that other large species differences may be masking effects of diet such as lifespan or biotransformation and elimination capacities. CP and dechlorane concentrations were higher than in other marine mammals from the (sub)Arctic, and they were present in a killer whale neonate, indicating bioaccumulative properties and a potential for maternal transfer in these predominantly unregulated chemicals.

Hormonal basis of seasonal metabolic changes in mammalian species.

Seasonal changes in external conditions (photoperiod, meteorological conditions, diet) cause adaptive changes in both energy and substrate metabolism in the animals of mammalian species. In summer, long days and a rich diet contribute to relative elevation in the levels of thyroid hormones (TH), but warmer weather lowers their levels. In winter, short days and a poor diet inhibit TH synthesis, but low temperatures increase their secretion. In addition, the results of our meta-analyses revealed a significant role of atmospheric pressure in circannual fluctuations of metabolic parameters in humans. The changes in photoperiod are generally viewed as a major factor contributing to seasonal rhythm regulation However, numerous data show that season-dependent metabolic changes in mammals could be also accounted for by meteorological factors and diet.

The mechanism of mammalian proton-coupled peptide transporters.

Proton-coupled oligopeptide transporters (POTs) are of great pharmaceutical interest owing to their promiscuous substrate binding site that has been linked to improved oral bioavailability of several classes of drugs. Members of the POT family are conserved across all phylogenetic kingdoms and function by coupling peptide uptake to the proton electrochemical gradient. Cryo-EM structures and alphafold models have recently provided new insights into different conformational states of two mammalian POTs, SLC15A1, and SLC15A2. Nevertheless, these studies leave open important questions regarding the mechanism of proton and substrate coupling, while simultaneously providing a unique opportunity to investigate these processes using molecular dynamics (MD) simulations. Here, we employ extensive unbiased and enhanced-sampling MD to map out the full SLC15A2 conformational cycle and its thermodynamic driving forces. By computing conformational free energy landscapes in different protonation states and in the absence or presence of peptide substrate, we identify a likely sequence of intermediate protonation steps that drive inward-directed alternating access. These simulations identify key differences in the extracellular gate between mammalian and bacterial POTs, which we validate experimentally in cell-based transport assays. Our results from constant-PH MD and absolute binding free energy (ABFE) calculations also establish a mechanistic link between proton binding and peptide recognition, revealing key details underpining secondary active transport in POTs. This study provides a vital step forward in understanding proton-coupled peptide and drug transport in mammals and pave the way to integrate knowledge of solute carrier structural biology with enhanced drug design to target tissue and organ bioavailability.

The cells in our body are sealed by a surrounding membrane that allows them to control which molecules can enter or leave. Desired molecules are often imported via transport proteins that require a source of energy. One way that transporter proteins achieve this is by simultaneously moving positively charged particles called protons across the membrane. Proteins called POTs (short for proton-coupled oligopeptide transporters) use this mechanism to import small peptides and drugsin to the cells of the kidney and small intestine. Sitting in the centre of these transporters is a pocket that binds to the imported peptide which has a gate on either side: an outer gate that opens towards the outside of the cell, and an inner gate that opens towards the cell's interior. The movement of protons from the outer to the inner gate is thought to shift the shape of the transporter from an outwards to an inwards-facing state. However, the molecular details of this energetic coupling are not well understood. To explore this, Lichtinger et al. used computer simulations to pinpoint where protons bind on POTs to trigger the gates to open. The simulations proposed that two sites together make up the outward-facing gate, which opens upon proton binding. Lichtinger et al. then validated these sites experimentally in cultured human cells that produce mutant POTs. After the desired peptide/drug has attached to the binding pocket, the protons then move to two more sites further down the transporter. This triggers the inner gate to open, which ultimately allows the small molecule to move into the cell. These findings represent a significant step towards understanding how POTs transport their cargo. Since POTs can transport a range of drugs from the digestive tract into the body, these results could help researchers design molecules that are better absorbed. This could lead to more orally available medications, making it easier for patients to adhere to their treatment regimen.

Screening 800 putative endocrine disrupting chemicals in a representative mammal, bird, and fish using a neurochemical cell-free testing platform.

There is global demand for novel ecotoxicity testing tools that are based on alternative to animal models, have high throughput potential, and may be applicable to a wide diversity of taxa. Here we scaled up a microplate-based cell-free neurochemical testing platform to screen 800 putative endocrine disrupting chemicals from the U.S. Environmental Protection Agency's ToxCast e1k library against the glutamate (NMDA), muscarinic acetylcholine (mACh), and dopamine (D2) receptors. Each assay was tested in cellular membranes isolated from brain tissues from a representative bird (zebra finch = Taeniopygia castanotis), mammal (mink = Neogale vison), and fish (rainbow trout = Oncorhynchus mykiss). The primary objective of this short communication was to make the results database accessible, while also summarising key attributes of assay performance and presenting some initial observations. In total, 7200 species-chemical-assay combinations were tested, of which 453 combinations were classified as a hit (radioligand binding changed by at least 3 standard deviations). There were some differences across species, and most hits were found for the D2 and NMDA receptors. The most active chemical was C.I. Solvent Yellow 14 followed by Diphenhydramine hydrochloride, Gentian Violet, SR271425, and Zamifenacin. Nine chemicals were tested across multiple plates with a mean relative standard deviation of the specific radioligand binding data being 24.6%. The results demonstrate that cell-free assays may serve as screening tools for large chemical libraries especially for ecological species not easily studied using traditional methods.

Size matters in metabolic scaling: Critical role of the thermodynamic efficiency of ATP synthesis and its dependence on mitochondrial H+ leak across mammalian species.

In this last article of the trilogy, the unified biothermokinetic theory of ATP synthesis developed in the previous two papers is applied to a major problem in comparative physiology, biochemistry, and ecology-that of metabolic scaling as a function of body mass across species. A clear distinction is made between intraspecific and interspecific relationships in energy metabolism, clearing up confusion that had existed from the very beginning since Kleiber first proposed his mouse-to-elephant rule almost a century ago. It is shown that the overall mass exponent of basal/standard metabolic rate in the allometric relationship [Formula: see text] is composed of two parts, one emerging from the relative intraspecific constancy of the slope (b), and the other (b') arising from the interspecific variation of the mass coefficient, a(M) with body size. Quantitative analysis is shown to reveal the hidden underlying relationship followed by the interspecific mass coefficient, a(M)=P0M0.10, and a universal value of P0=3.23 watts, W is derived from empirical data on mammals from mouse to cattle. The above relationship is shown to be understood only within an evolutionary biological context, and provides a physiological explanation for Cope's rule. The analysis also helps in fundamentally understanding how variability and a diversity of scaling exponents arises in allometric relations in biology and ecology. Next, a molecular-level understanding of the scaling of metabolism across mammalian species is shown to be obtained by consideration of the thermodynamic efficiency of ATP synthesis η, taking mitochondrial proton leak as a major determinant of basal metabolic rate in biosystems. An iterative solution is obtained by solving the mathematical equations of the biothermokinetic ATP theory, and the key thermodynamic parameters, e.g. the degree of coupling q, the operative P/O ratio, and the metabolic efficiency of ATP synthesis η are quantitatively evaluated for mammals from rat to cattle. Increases in η (by ∼15%) over a 2000-fold body size range from rat to cattle, primarily arising from an ∼3-fold decrease in the mitochondrial H+ leak rate are quantified by the unified ATP theory. Biochemical and mechanistic consequences for the interpretation of basal metabolism, and the various molecular implications arising are discussed in detail. The results are extended to maximum metabolic rate, and interpreted mathematically as a limiting case of the general ATP theory. The limitations of the analysis are pointed out. In sum, a comprehensive quantitative analysis based on the unified biothermokinetic theory of ATP synthesis is shown to solve a central problem in biology, physiology, and ecology on the scaling of energy metabolism with body size.

Circadian-independent light regulation of mammalian metabolism.

The daily light-dark cycle is a key zeitgeber (time cue) for entraining an organism's biological clock, whereby light sensing by retinal photoreceptors, particularly intrinsically photosensitive retinal ganglion cells, stimulates the suprachiasmatic nucleus of the hypothalamus, a central pacemaker that in turn orchestrates the rhythm of peripheral metabolic activities. Non-rhythmic effects of light on metabolism have also been long known, and their transduction mechanisms are only beginning to unfold. Here, we summarize emerging evidence that, in mammals, light exposure or deprivation profoundly affects glucose homeostasis, thermogenesis and other metabolic activities in a clock-independent manner. Such light regulation could involve melanopsin-based, intrinsically photosensitive retinal ganglion cell-initiated brain circuits via the suprachiasmatic nucleus of the hypothalamus and other nuclei, or direct stimulation of opsins expressed in the hypothalamus, adipose tissue, blood vessels and skin to regulate sympathetic tone, lipolysis, glucose uptake, mitochondrial activation, thermogenesis, food intake, blood pressure and melanogenesis. These photic signalling events may coordinate with circadian-based mechanisms to maintain metabolic homeostasis, with dysregulation of this system underlying metabolic diseases caused by aberrant light exposure, such as environmental night light and shift work.

Cytosolic and Acrosomal pH Regulation in Mammalian Sperm.

As in most cells, intracellular pH regulation is fundamental for sperm physiology. Key sperm functions like swimming, maturation, and a unique exocytotic process, the acrosome reaction, necessary for gamete fusion, are deeply influenced by pH. Sperm pH regulation, both intracellularly and within organelles such as the acrosome, requires a coordinated interplay of various transporters and channels, ensuring that this cell is primed for fertilization. Consistent with the pivotal importance of pH regulation in mammalian sperm physiology, several of its unique transporters are dependent on cytosolic pH. Examples include the Ca2+ channel CatSper and the K+ channel Slo3. The absence of these channels leads to male infertility. This review outlines the main transport elements involved in pH regulation, including cytosolic and acrosomal pH, that participate in these complex functions. We present a glimpse of how these transporters are regulated and how distinct sets of them are orchestrated to allow sperm to fertilize the egg. Much research is needed to begin to envision the complete set of players and the choreography of how cytosolic and organellar pH are regulated in each sperm function.

Mammalian pexophagy at a glance.

Peroxisomes are highly plastic organelles that are involved in several metabolic processes, including fatty acid oxidation, ether lipid synthesis and redox homeostasis. Their abundance and activity are dynamically regulated in response to nutrient availability and cellular stress. Damaged or superfluous peroxisomes are removed mainly by pexophagy, the selective autophagy of peroxisomes induced by ubiquitylation of peroxisomal membrane proteins or ubiquitin-independent processes. Dysregulated pexophagy impairs peroxisome homeostasis and has been linked to the development of various human diseases. Despite many recent insights into mammalian pexophagy, our understanding of this process is still limited compared to our understanding of pexophagy in yeast. In this Cell Science at a Glance article and the accompanying poster, we summarize current knowledge on the control of mammalian pexophagy and highlight which aspects require further attention. We also discuss the role of ubiquitylation in pexophagy and describe the ubiquitin machinery involved in regulating signals for the recruitment of phagophores to peroxisomes.

Exploring Fundamentals of Prion Biology Using Natural Yeast Prions and Mammalian PrP.

The key postulate of the prion paradigm is that some proteins can take on unconventional conformations and pass these conformations to newly synthesized protein molecules with the same primary structure [...].

Reproductive Ageing: Metabolic contribution to age-related chromosome missegregation in mammalian oocytes.

In brief: Chromosome missegregation and declining energy metabolism are considered to be unrelated features of oocyte ageing that contribute to poor reproductive outcomes. Given the bioenergetic cost of chromosome segregation, we propose here that altered energy metabolism during ageing may be an underlying cause of age-related chromosome missegregation and aneuploidy. Abstract: Advanced reproductive age in women is a major cause of infertility, miscarriage and congenital abnormalities. This is principally caused by a decrease in oocyte quality and developmental competence with age. Oocyte ageing is characterised by an increase in chromosome missegregation and aneuploidy. However, the underlying mechanisms of age-related aneuploidy have not been fully elucidated and are still under active investigation. In addition to chromosome missegregation, oocyte ageing is also accompanied by metabolic dysfunction. In this review, we integrate old and new perspectives on oocyte ageing, chromosome segregation and metabolism in mammalian oocytes and make direct links between these processes. We consider age-related alterations to chromosome segregation machinery, including the loss of cohesion, microtubule stability and the integrity of the spindle assembly checkpoint. We focus on how metabolic dysfunction in the ageing oocyte disrupts chromosome segregation machinery to contribute to and exacerbate age-related aneuploidy. More specifically, we discuss how mitochondrial function, ATP production and the generation of free radicals are altered during ageing. We also explore recent developments in oocyte metabolic ageing, including altered redox reactions (NAD+ metabolism) and the interactions between oocytes and their somatic nurse cells. Throughout the review, we integrate the mechanisms by which changes in oocyte metabolism influence age-related chromosome missegregation.

Energetic cost of biosynthesis is a missing link between growth and longevity in mammals.

The comparative studies of aging have established a negative correlation between Gompertz postnatal growth constant and maximum lifespan across mammalian species, but the underlying physiological mechanism remains unclear. This study shows that the Gompertz growth constant can be decomposed into two energetic components, mass-specific metabolic rate and the energetic cost of biosynthesis, and that after controlling the former as a confounder, the negative correlation between growth constant and lifespan still exists due to a 100-fold variation in the latter, revealing that the energetic cost of biosynthesis is a link between growth and longevity in mammals. Previously, the energetic cost of biosynthesis has been thought to be a constant across species and therefore was not considered a contributor to the variation in any life history traits, such as growth and lifespan. This study employs a recently proposed model based on energy conservation to explain the physiological effect of the variation in this energetic cost on the aging process and illustrates its role in linking growth and lifespan. The conventional life history theory suggested a tradeoff between growth and somatic maintenance, but the findings in this study suggest that allocating more energy to biosynthesis may enhance the somatic maintenance and extend lifespan and, hence, reveal a more complex nature of the tradeoff.

No evidence for a signal in mammalian basal metabolic rate associated with a fossorial lifestyle.

A vast array of challenging environments are inhabited by mammals, such as living in confined spaces where oxygen levels are likely to be low. Species can exhibit adaptations in basal metabolic rate (BMR) to exploit such unique niches. In this study we use 801 species to determine the relationship between BMR and burrow use in mammals. We included pre-existing data for mammalian BMR and 16 life history traits. Overall, mammalian BMR is dictated primarily by environmental ambient temperature. There were no significant differences in BMR of terrestrial, semi-fossorial and fossorial mammals, suggesting that species occupying a subterranean niche do not exhibit baseline metabolic costs on account of their burrowing lifestyle. Fossorial mammals likely show instantaneous metabolic responses to low oxygen in tunnels, rather than exhibit adaptive long-term responses in their BMR.

Neuropeptides affecting social behavior in mammals: Oxytocin.

Oxytocin (OXT), a neuropeptide consisting of only nine amino acids, is synthesized in the paraventricular and supraoptic nuclei of the hypothalamus. Although OXT is best known for its role in lactation and parturition, recent research has shown that it also has a significant impact on social behaviors in mammals. However, a comprehensive review of this topic is still lacking. In this paper, we systematically reviewed the effects of OXT on social behavior in mammals. These effects of OXT from the perspective of five key behavioral dimensions were summarized: parental behavior, anxiety, aggression, attachment, and empathy. To date, researchers have agreed that OXT plays a positive regulatory role in a wide range of social behaviors, but there have been controversially reported results. In this review, we have provided a detailed panorama of the role of OXT in social behavior and, for the first time, delved into the underlying regulatory mechanisms, which may help better understand the multifaceted role of OXT. Levels of OXT in previous human studies were also summarized to provide insights for diagnosis of mental disorders.

Early embryonic polarity establishment and implications for lineage differentiation.

Polarity establishment is one of the key factors affecting early embryonic development. Polarity establishment begins with myosin phosphorylation in the 8-cell embryo, and phosphorylation activates actin leading to its initiation of contractility. Subsequently, actin undergoes reorganization to form an apical domain rich in microvilli on the non-contacting surface of each blastomere, and form the actomyosin ring that marks the maturation of the apical domain in conjunction with polar protein complexes and others. From the process of polarity establishment, it can be seen that the formation of the apical domain is influenced by actin-related proteins and polar protein complexes. Some zygote genome activation (ZGA) and lineage-specific genes also regulate polarity establishment. Polarity establishment underlies the first cell lineage differentiation during early embryonic development. It regulates lineage segregation and morphogenesis by affecting asymmetric cell division, asymmetric localization of lineage differentiation factors, and activity of the Hippo signaling pathway. In this review, we systematically summarize the mechanisms of early embryonic polarity establishment and its impact on lineage differentiation in mammals, and discuss the shortcomings of the currently available studies in terms of regulatory mechanisms and species, thereby providing clues and systematic perspectives for elucidating early embryonic polarity establishment.

Immunolabeling for filaggrin and acidic keratins in the granular layer of mammalian epidermis indicates that an acidic-basic interaction is involved in cornification.

The soft epidermis of mammals derives from the accumulation of keratohyaline granules in the granular layer, before maturing into corneocytes. Main proteins accumulated in the granular layer are pro-filaggrin and filaggrin that determine keratin clumping and later moisturization of the stratum corneum that remains flexible. This soft epidermis allows the high sensitivity of mammalian skin. Presence and thickness of the stratum granulosum varies among different species of mammals and even between different body regions of the same animal, from discontinuous to multilayered. These variations are evident using antibodies for filaggrin, a large protein that share common epitopes among placentals. Here we have utilized filaggrin antibodies (8959 and 466) and an acidic keratin antibody (AK2) for labeling placental, marsupial and monotreme epidermis. AK2 labeling appears mainly to detect K24 keratin, and less likely other acidic keratins. Immunoreactivity for filaggrin is absent in platypus, discontinuous in Echidna and in the tested marsupials. In placentals, it is inconstantly or hardly detected in the thin epidermis of bat, rodents, and lagomorphs with a narrow, mono-stratified and/or discontinuous granular layer. In contrast, where the granular layer is continuous or even stratified, both filaggrin and AK2 antibodies decorate granular cells. The ultrastructural analysis using the AK2 antibody on human epidermis reveals that a weak labeling is associated with keratohyalin granules and filamentous keratins of transitional keratinocytes and corneocytes. This observation suggests that basophilic filaggrin interacts with acidic keratins like K24 and determines keratin condensation into corneocytes of the stratum corneum.

Ubiquitinome Analysis Uncovers Alterations in Synaptic Proteins and Glucose Metabolism Enzymes in the Hippocampi of Adolescent Mice Following Cold Exposure.

Cold exposure exerts negative effects on hippocampal nerve development in adolescent mice, but the underlying mechanisms are not fully understood. Given that ubiquitination is essential for neurodevelopmental processes, we attempted to investigate the effects of cold exposure on the hippocampus from the perspective of ubiquitination. By conducting a ubiquitinome analysis, we found that cold exposure caused changes in the ubiquitination levels of a variety of synaptic-associated proteins. We validated changes in postsynaptic density-95 (PSD-95) ubiquitination levels by immunoprecipitation, revealing reductions in both the K48 and K63 polyubiquitination levels of PSD-95. Golgi staining further demonstrated that cold exposure decreased the dendritic-spine density in the CA1 and CA3 regions of the hippocampus. Additionally, bioinformatics analysis revealed that differentially ubiquitinated proteins were enriched in the glycolytic, hypoxia-inducible factor-1 (HIF-1), and 5'-monophosphate (AMP)-activated protein kinase (AMPK) pathways. Protein expression analysis confirmed that cold exposure activated the mammalian target of rapamycin (mTOR)/HIF-1α pathway. We also observed suppression of pyruvate kinase M2 (PKM2) protein levels and the pyruvate kinase (PK) activity induced by cold exposure. Regarding oxidative phosphorylation, a dramatic decrease in mitochondrial respiratory-complex I activity was observed, along with reduced gene expression of the key subunits NADH: ubiquinone oxidoreductase core subunit V1 (Ndufv1) and Ndufv2. In summary, cold exposure negatively affects hippocampal neurodevelopment and causes abnormalities in energy homeostasis within the hippocampus.

An Automated Imaging-Based Screen for Genetic Modulators of ER Organisation in Cultured Human Cells.

Hereditary spastic paraplegias (HSPs) are a heterogeneous group of mono-genetic inherited neurological disorders, whose primary manifestation is the disruption of the pyramidal system, observed as a progressive impaired gait and leg spasticity in patients. Despite the large list of genes linked to this group, which exceeds 80 loci, the number of cellular functions which the gene products engage is relatively limited, among which endoplasmic reticulum (ER) morphogenesis appears central. Mutations in genes encoding ER-shaping proteins are the most common cause of HSP, highlighting the importance of correct ER organisation for long motor neuron survival. However, a major bottleneck in the study of ER morphology is the current lack of quantitative methods, with most studies to date reporting, instead, on qualitative changes. Here, we describe and apply a quantitative image-based screen to identify genetic modifiers of ER organisation using a mammalian cell culture system. An analysis reveals significant quantitative changes in tubular ER and dense sheet ER organisation caused by the siRNA-mediated knockdown of HSP-causing genes ATL1 and RTN2. This screen constitutes the first attempt to examine ER distribution in cells in an automated and high-content manner and to detect genes which impact ER organisation.