Biology subjects

Newman, J. C.

Publications and source records attributed to Newman, J. C..

12 recordsLinked to original sources

bioRxiv2026

Advanced Open-source Experimental-Design Tools for Microplate-Based Assays with Acoustic Liquid Handling

Acoustic droplet ejection (ADE) enables nanoliter-scale liquid handling for complex microplate assays, yet translating experimental designs into validated, instrument-ready instructions remains a bottleneck. We present PickliPy, an open-source framework that converts spreadsheet-based assay designs into validated ADE picklists. PickliPy.Assay supports combinatorial, dose-response, and multi-addition time-course dispensing, while PickliPy.Screen extends to high-throughput workflows, including library reformatting and shortlisting. Across biological contexts, the framework generated reproducible, assay-ready plates and standardized execution in human cohort studies. Acoustic pre-dispensing deepened bioenergetic phenotyping of human skeletal muscle mitochondria, capturing substrate switching and sharpened dose-response precision in human pancreatic {beta}-cells, revealing an age-associated change in succinate dehydrogenase kinetics. We benchmarked a wash-free, live-cell screen of mitochondrial function and morphology, in which deep-learning image analysis widened the assay window, and ADE enabled integrative dose-response co-response analysis. These tools, including their agentic use, make complex ADE experiments easier to design and scale from single benches to screening campaigns.

cell biology↗

bioRxiv2026

Ketone ester supplementation in aged mice reduces activation of B cell subsets

BackgroundAging in the immune system results in increased susceptibility to infections, exacerbated autoimmunity, and reduced responsiveness to vaccines. However, there are no current established interventions for immune aging. Ketogenic diets and fasting have been researched as interventions against other aspects of aging and age-related diseases, and they work in part by increasing circulating levels of ketone bodies, which have anti-inflammatory properties and can boost T cell function. Exogenous ketones, such as ketone esters, are currently being studied as a more accessible approach to obtain the benefits of ketone bodies through direct supplementation. Here, we investigated whether ketone ester supplementation improves immune function during aging. Aged (19-month-old) C57BL/6JN mice were given a diet supplemented with the ketone ester or a control diet for 15 weeks. ResultsWe found that the ketone ester diet decreased activation of B cells, especially age-associated B cells, in the spleen. The diet also reduced the proportion of age-associated B cells defined by CD11b and CD11c expression, though not of the broader CD21-CD23- population. Despite this decrease in activation, there was no impairment in antibody production, as mice on the ketone ester diet mounted a normal response to nitrophenyl-ovalbumin immunization. The ketone ester diet also reduced glucose dependence and translation in subsets of age-associated B cells. Treating splenocytes from aged mice with {beta}-hydroxybutyrate was sufficient to suppress B cell translation and reduced mTORC1 signaling, indicating that ketone bodies act directly on B cells. {beta}-hydroxybutyrate also decreased expression of T-bet, the signature transcription factor driving the autoimmune functions of age-associated B cells. ConclusionsOur study elucidates the effect of ketone esters on B cells in the context of aging and unveils a new immunoregulatory role of ketone bodies on B cells.

immunology↗

bioRxiv2026

Requirement for oxidation of neuronal ketone bodies in aging andneurodegeneration

Glucose is the brains primary fuel, but the brain can also use alternative energy substrates, especially during development or starvation. Emerging evidence suggests ketone metabolism may help the brain adapt to energy stress in neurodegenerative diseases such as Alzheimers disease, although its role in constitutive brain function in normal aging is poorly understood. Using iPSC-derived human neurons and adult-inducible, neuron-specific Bdh1 knockout mice, we show that ketone body metabolism is essential for maximum energy production, neuronal function, and mouse survival--even under normal nutritional conditions. Mechanistically, phenotypes of Bdh1 knockout neurons are mitigated by provision of acetoacetate, a downstream energy metabolite. Moreover, loss of neuronal ketone oxidation markedly increases mortality and memory deficits in Alzheimers disease model mice. These findings identify ketones as critical neuronal fuels, with particular importance during neurodegeneration. While non-energetic activities of ketone bodies are increasingly appreciated, oxidation for energy provision is an essential mechanism for normal function in neurons and mice. Targeting the energetic function of ketones may thus offer new therapeutic strategies for both aging and neurodegenerative diseases such as Alzheimers.

neuroscience↗

bioRxiv2026

Ketone body beta-hydroxybutyrate restores neuronal Tau proteostasis via ketolysis-independent mechanism

Metabolic interventions that induce ketosis, including ketogenic diets, caloric restriction, intermittent fasting, and exercise, show promise in the treatment of Alzheimers disease (AD) and related tauopathies. {beta}-hydroxybutyrate ({beta}HB), the primary ketone body produced during ketosis, reproduces key features of these metabolic interventions, but the molecular mechanism underlying its neuroprotective properties is not fully understood. Here, we demonstrate that a {beta}HB precursor diet is sufficient to ameliorate Tau pathophysiology in a tauopathy mouse model. Furthermore, across in vitro, ex vivo, and in vivo models, we find that {beta}HB enhances neuronal Tau proteostasis and reduces Tau aggregation and secretion. Importantly, these effects are independent of {beta}HBs oxidation for ATP production, as its ketolysis-resistant enantiomer reproduces these benefits, indicating that ketolysis is dispensable for these effects. Overall, these data position {beta}HB as a novel therapeutic avenue for AD and tauopathy and elucidate a novel mechanism of action of metabolic interventions in neurodegenerative disease.

neuroscience↗

bioRxiv2026

Ketone bodies mitigate against systemic inflammation-induced changes in brain energy metabolism and delirium-like deficits in aged mice

Acute systemic inflammation affects brain function, with detrimental consequences in aged individuals. These include delirium, an acute neuropsychiatric syndrome characterized by fluctuating disturbances in attention, perception and cognition. Delirium is associated with disrupted brain energy metabolism but our understanding of this during acute systemic inflammation is limited. Here we hypothesized that LPS-induced systemic inflammation would disrupt brain energy metabolism in aged C57BL6J mice and that the consequent functional impairments would be mitigated by ketone body utilization. We investigated ketone body effects in sickness behaviour, inflammation, energy metabolism and cognitive function. Real-time changes in utilisation of energy sources were quantified by indirect calorimetry and administration of radioisotope-labelled glucose and betahydroxybutyrate. Mass-spectrometry metabolomics was used to index severity of behavioural distrurbances to changes in hippocampal energy metabolism. LPS precipitated hypoglycemia and induced a whole-body switch from carbohydrate to lipid utilisation. Despite this, hippocampal insulin resistance and preserved brain glucose was observed while alternative carbohydrates, mannose and fructose, became depleted. Ketone ester treatment reversed insulin resistance, mitigated sickness behaviour and prevented delirium-like cognitive dysfunction without altering pro-inflammatory responses. Our results show that promoting ketone body usage mitigates systemic inflammation-induced brain energy disruption and prevents delirium-like cognitive deficits in aged mice.

neuroscience↗

bioRxiv2023

A ketogenic diet reduces age-induced chronic neuroinflammation in mice

Beta-hydroxybutyrate (BHB) is a ketone body synthesized during fasting or strenuous exercise. Our previous study demonstrated that a cyclic ketogenic diet (KD), which induces BHB levels similar to fasting every other week, reduces midlife mortality and improves memory in aging mice. BHB actively regulates gene expression and inflammatory activation through non-energetic signaling pathways. Neither of these activities has been well-characterized in the brain and they may represent mechanisms by which BHB affects brain function during aging. First, we analyzed hepatic gene expression in an aging KD-treated mouse cohort using bulk RNA-seq. In addition to the downregulation of TOR pathway activity, cyclic KD reduces inflammatory gene expression in the liver. We observed via flow cytometry that KD also modulates age-related systemic T cell functions. Next, we investigated whether BHB affects brain cells transcriptionally in vitro. Gene expression analysis in primary human brain cells (microglia, astrocytes, neurons) using RNA-seq shows that BHB causes a mild level of inflammation in all three cell types. However, BHB inhibits the more pronounced LPS-induced inflammatory gene activation in microglia. Furthermore, we confirmed that BHB similarly reduces LPS-induced inflammation in primary mouse microglia and bone marrow-derived macrophages (BMDMs). BHB is recognized as an inhibitor of histone deacetylase (HDAC), an inhibitor of NLRP3 inflammasome, and an agonist of the GPCR Hcar2. Nevertheless, in microglia, BHBs anti-inflammatory effects are independent of these known mechanisms. Finally, we examined the brain gene expression of 12-month-old male mice fed with one-week and one-year cyclic KD. While a one-week KD increases inflammatory signaling, a one-year cyclic KD reduces neuroinflammation induced by aging. In summary, our findings demon-strate that BHB mitigates the microglial response to inflammatory stimuli, like LPS, possibly leading to decreased chronic inflammation in the brain after long-term KD treatment in aging mice.

immunology↗

bioRxiv2023

A diet-dependent host metabolite shapes the gut microbiota to protect from autoimmunity

Diet can protect from autoimmune disease; however, whether diet acts via the host and/or microbiome remains unclear. Here, we use a ketogenic diet (KD) as a model to dissect these complex interactions. A KD rescued the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis in a microbiota-dependent fashion. Dietary supplementation with a single KD-dependent host metabolite ({beta}-hydroxybutyrate, {beta}HB) rescued EAE whereas transgenic mice unable to produce {beta}HB in the intestine developed more severe disease. Transplantation of the {beta}HB-shaped gut microbiota was protective. Lactobacillus sequence variants were associated with decreased T helper 17 (Th17) cell activation in vitro. Finally, we isolated a L. murinus strain that protected from EAE, which was phenocopied by the Lactobacillus metabolite indole lactic acid. Thus, diet alters the immunomodulatory potential of the gut microbiota by shifting host metabolism, emphasizing the utility of taking a more integrative approach to study diet-host-microbiome interactions.

microbiology↗

bioRxiv2023

Ketogenic diet administration later in life improves memory and regulates the synaptic cortical proteome through the cAMP/PKA signaling pathway in aging mice.

Aging is a complex biological process that compromises brain function and neuronal network activity, leading to cognitive decline and synaptic dysregulation. In recent years, a cyclic Ketogenic Diet (KD) has emerged as a potential treatment to ameliorate cognitive decline by improving memory in aged mice after long-term administration. However, the molecular mechanisms that govern such changes remains unclear. Additionally, whether short-term cyclic KD administration later in life preserves memory has not been addressed in detail. Accordingly, here we investigated how a short-term cyclic KD starting at 20-23 months-old regulates brain function of aged mice. Behavioral testing and long-term potentiation (LTP) recordings revealed that a cyclic KD improves working memory and hippocampal LTP in 24-27 months-old mice after 16 weeks of treatment. Moreover, the diet also promotes higher dendritic arborization complexity and dendritic spine density in the prefrontal cortex. Furthermore, to elucidate the molecular mechanisms underlying the memory improvements elicited by a cyclic KD, cortical synaptosomes of aged mice fed with this diet for 1 year were analyzed by mass spectrometry. Bioinformatics analysis revealed that long-term cyclic KD administration predominantly modulates the presynaptic compartment by inducing changes in the cAMP/PKA signaling pathway, the synaptic vesicle cycle, and the actin/microtubule cytoskeleton. To test these findings in vivo, synaptic proteins from cortices of 24-27-month-old mice fed with control or cyclic KD for 16 weeks were analyzed by western blot. Interestingly, increased Brain Derived Neurotrophic Factor abundance, MAP2 phosphorylation and PKA activity were observed. Overall, we show that a cyclic KD regulates brain function and memory even when it is administered at late mid-life and significantly triggers several molecular features of long-term administration, including the PKA signaling pathway and cytoskeleton dynamics, thus promoting synaptic plasticity at advanced age.

neuroscience↗

bioRxiv2023

β-hydroxybutyrate is a metabolic regulator of proteostasis in the aged and Alzheimer disease brain

Loss of proteostasis is a hallmark of aging and Alzheimer disease (AD). Here, we identify {beta}-hydroxybutyrate ({beta}HB), a ketone body, as a regulator of protein solubility in the aging brain. {beta}HB is a small molecule metabolite which primarily provides an oxidative substrate for ATP during hypoglycemic conditions, and also regulates other cellular processes through covalent and noncovalent protein interactions. We demonstrate {beta}HB-induced protein insolubility across in vitro, ex vivo, and in vivo mouse systems. This activity is shared by select structurally similar metabolites, is not dependent on covalent protein modification, pH, or solute load, and is observable in mouse brain in vivo after delivery of a ketone ester. Furthermore, this phenotype is selective for pathological proteins such as amyloid-{beta}, and exogenous {beta}HB ameliorates pathology in nematode models of amyloid-{beta} aggregation toxicity. We have generated a comprehensive atlas of the {beta}HB-induced protein insolublome ex vivo and in vivo using mass spectrometry proteomics, and have identified common protein domains within {beta}HB target sequences. Finally, we show enrichment of neurodegeneration-related proteins among {beta}HB targets and the clearance of these targets from mouse brain, likely via {beta}HB-induced autophagy. Overall, these data indicate a new metabolically regulated mechanism of proteostasis relevant to aging and AD.

physiology↗

bioRxiv2022

Ketone body metabolism declines with age in mice in a sex-dependent manner

Understanding how our cells maintain energy homeostasis has long been a focus of aging biology. A decline in energy metabolism is central to many age-related diseases such as Alzheimers disease, heart failure, frailty, and delirium. Intervening on pathways involved in energy homeostasis can extend healthy lifespan. When the primary energy substrate glucose is scarce, mice and humans can partially switch cellular energetic needs to fat-derived ketone bodies (i.e., beta-hydroxybutyrate (BHB), acetoacetate, acetone). Aging is associated with glucose intolerance and insulin insensitivity, yet, surprisingly, what role ketone body metabolism might play in compensating for impaired glucose utilization in age-related diseases is understudied. Here, we investigate how endogenous ketone body production and utilization pathways are modulated by age across the lifespan of male and female C57BL/6N mice (3 mo old, 12 mo old, 22 mo old). We show how different ages have different metabolic and gene expression responses to 1-week ketogenic diet (KD). We hypothesized that there would be a compensatory ketogenic response with age but instead saw declines in plasma BHB concentrations under fasting and non-fasting conditions with strong sexual dimorphism. Under KD, both sexes increased BHB concentrations at all ages, but only males showed strong gene expression induction. We also observed tissue-specific changes with age in baseline ketone metabolism, and surprising induction of extrahepatic ketogenic genes under KD. We found significant residual blood concentrations of BHB in KD even after a knockout of liver BHB production. Overall, these findings show that older mice have impaired non-fasting ketogenesis but are capable of increasing their ketogenic capacity under stimulation (i.e., KD) to meet energetic demands in aging. Therapies to augment non-fasting ketogenesis or provide exogenous ketones may be useful to improve energy homeostasis in diseases and conditions of aging.

physiology↗

bioRxiv2022

Small-molecule ketone esters treat brain network abnormalities in an Alzheimer's disease mouse model

Altered brain network activity and the resulting hypersynchrony are important for the pathogenesis of cognitive decline in Alzheimers disease (AD) mouse models. Treatments that reduce epileptiform discharges (EDs) or network hyperactivity improve cognition in AD models and humans. We first show that ketogenic diet, but not fasting, rapidly and persistently reduced EDs in the hAPPJ20 Alzheimers mouse model over timescales of hours to months. Then, to identify the specific mechanism of the pleiotropic ketogenic diet, we developed small molecule ketone esters to deliver ketone bodies pharmacologically. Two ketone esters recapitulate ED suppression without other dietary manipulation, over time scales of minutes to one week. This small molecule rescue was associated with reduced low-frequency oscillatory activity similar to the recently reported mechanism of an NMDA receptor modulator molecule in this model. Long-term KD resulted in cognitive improvement and in a sex-stratified analysis also improved survival in the more severely affected hAPPJ20 males. Agents that deliver ketone bodies via small molecules or act on downstream targets may hold therapeutic promise in AD through the mechanism of improved network function and reduced epileptiform activity.

neuroscience↗

bioRxiv2017

Ketogenic diet or BHB improves epileptiform spikes, memory, survival in Alzheimer’s model

Links between epilepsy and Alzheimers disease (AD) are seen in both human patients and mouse models. Human patients with AD may commonly have subclinical epileptiform spikes (EP spikes)1, and overt epilepsy is associated with more rapid cognitive decline2. Mechanistic studies in mouse models of Alzheimers disease (AD) have shown that altered network activity and epileptiform spikes stem from dysfunctional inhibitory interneurons3, which are key elements of cortical circuits underlying cognition4. Treatments that reduce epileptiform spikes improve cognition in these models5,6. Thus, targeting subclinical epileptiform activity may be a promising new therapeutic approach to AD7. Ketogenic diet (KD) has long been used to treat forms of epilepsy8, including Dravet syndrome, a childhood epilepsy caused by mutations in a gene that is critical for inhibitory interneuron function in mouse AD models5,9. However, the concurrent effects of a ketogenic diet on brain electrical activity, cognitive decline, and survival have not been tested, and the translational rationale and feasibility of such an intervention remain uncertain. Here we show that a ketogenic diet reduces epileptiform spikes in the hAPPJ20 mouse model of AD. Similar reduction of EP spikes is observed using a {beta}-hydroxybutyrate (BHB) ester in both AD and Dravet mice. A ketogenic diet improves context-dependent and visuo-spatial learning in hAPPJ20 mice. It also reduces the high seizure-related mortality observed in male mice of this model. Therapies derived from {beta}-hydroxybutyrate may have potential application in ameliorating cognitive dysfunction in AD through reducing subclinical epileptiform activity.

neuroscience↗