Discovery of bioactive phytochemicals from botanical extracts is a pivotal step for the modernization of traditional medicine and in the discovery of novel pharmaceuticals. However, traditional bioassay-guided fractionation is laborious and often leads to the re-identification of known compounds. To address these challenges, we have developed a novel discovery pipeline, called CERES (Cluster-Enabled Regression of Extract Signatures) that combines preparative column chromatography-based fractionation and bioactivity assessment, followed by loop-injection ion-mobility mass spectrometry, with machine learning. We apply cluster-based feature reduction and regularized regression models for rapid identification of bioactive phytochemicals. This approach reduces the need for analytical chromatographic separation in early dereplication stages, reducing bias and increasing speed, while remaining complementary to downstream structural confirmation workflows. By grouping mass spectrometry features into clusters representing different chemical entities derived from the same molecular species, the computational approach effectively reduces dimensionality and improves performance and interpretability of model results. The computational approach was successfully applied here to identify bioactive compounds in fractions of Centella asiatica water extract and uncovered bioactive mass-to-charge signals linked to known chemical features as well as new low-abundant, yet to be annotated signals to be investigated in further studies. This innovative approach offers a powerful and efficient way to advance the integration of traditional medicine with evidence-based practice by leveraging the power of ion-mobility mass spectrometry and machine learning.

Heterozygote carriers of Gaucher's disease mutations and other polymorphisms in the glucocerebrosidase (GBA) gene show an increased incidence of Parkinson's disease. We hypothesized that common GBA polymorphisms would be associated with subtle parkinsonian features, mild cognitive impairment, and “silent” Lewy body (LB) pathology in aging individuals without a clinical diagnosis of parkinsonism. The most prevalent GBA variants, T369M and E326K, appear in the general population at rates of approximately 0.6% and 1%, respectively. We evaluated 845 participants from the Oregon Alzheimer's Disease Research Center (OADRC) with SNP data generated by the National Centralized Repository of Alzheimer's Disease (NCRAD). Twenty-one subjects were E326K carriers and eighteen were T369M carriers. Clinical measures and postmortem neuropathology were compared between each SNP group and non-carriers. Although there were no statistically significant clinical differences related to synucleinopathy across groups, neuropathological analyses revealed a significantly higher prevalence of LB pathology in E326K carriers compared to T369M carriers. When stratifying each genetic group by LB status (LB+ or LB−), LB+ E326K carriers demonstrated a significant reduction in Mini-Mental State Examination (MMSE) scores compared with LB− non-carriers and a modest decrease compared with T369M carriers. These preliminary findings from a small, uni-center cohort suggest that the E326K GBA polymorphism may predict LB pathology and subtle cognitive decline in aging individuals who lack overt parkinsonian symptoms. Further validation in a larger cohort is warranted. Identifying at-risk individuals through targeted genetic screening may ultimately support earlier intervention and preventative care strategies.

BACKGROUND: Inflammation is an early event that substantially influences Alzheimers disease (AD) pathogenesis, making it a compelling target for therapeutic intervention. Sob-AM2 is a brain-penetrating thyromimetic drug capable of inducing the expression of microglial cell surface receptor TREM2, which mediates the switch from pro-inflammatory to a more restorative microglial state. OBJECTIVE: Evaluate the effects of Sob-AM2 on cognition, AD pathology and microglial activity in the 5xFAD mouse model of amyloid-beta (Aβ) accumulation. METHODS: Seven-month-old 5xFAD mice and their wild-type littermates were administered Sob-AM2 subcutaneously three times per week for 12 weeks. In the last two weeks of treatment mice underwent behavioral tests to assess cognition and monitor for off-target mobility effects. At the end of treatment, brain tissue was harvested for gene and protein expression analyses. RESULTS: Sob-AM2 treatment increased TREM2 expression in the brains of 5xFAD mice. This was accompanied by an improvement in both spatial and associative memory as well as an increase in the expression of synaptic genes synaptophysin and PSD-95. No significant changes were detected in Aβ plaque burden or the expression of microglial activation marker Iba1 in Sob-AM2 treated animals, however, the expression of the phagocytic marker CD68 was significantly increased in the hippocampus, but not the cortex, in Sob-AM2 treated 5xFAD mice. CONCLUSION: These results suggest that the cognitive-enhancing effects of Sob-AM2 are not the result of reduced overall plaque burden. Future work is needed to further investigate the neuroprotective mechanism of Sob-AM2 and how it may be affecting microglial phenotypes.

Parkinson's Disease (PD) is the second most diagnosed neurological disorder globally, affecting millions of people worldwide. Oxidative stress is implicated in the progression of PD, yet its direct effects on motor function, particularly in the context of synucleinopathy, are not fully understood. Here, we investigated the effects of the loss of the antioxidant regulatory transcription factor NRF2 in the A53TSyn mouse model of synucleinopathy. Motor function was evaluated in separate cohorts of A53TSyn mice without NRF2 (A53TSyn/NRF2KO), as well as A53TSyn mice expressing NRF2 (A53TSyn/NRF2+) and healthy wild-type (WT) mice at four, six, and eight months of age. The overall mobility decreased in A53TSyn/NRF2KO mice relative to WT mice at all ages. Significant alterations in gait were also apparent in A53TSyn/NRF2KO mice compared to A53TSyn mice without NRF2 deletion. Expression of tyrosine hydroxylase (TH) was also quantified in the brains of those mice. While there were no differences in cortical pSyn expression between A53TSyn/NRF2+ and A53TSyn/NRF2KO mice, a reduction in TH abundance in the striatum was evident in A53TSyn/NRF2KO mice at all ages. In summary, our data suggest that NRF2 plays a role in maintaining mobility and gait in the context of synucleinopathy and may represent a therapeutic target to mitigate mobility decline in PD-affected individuals.

IntroductionSTX is a synthetic non-steroidal estrogen receptor modulator (SERM) that can provide many of the beneficial effects of 17β-estradiol in the brain without its adverse side effects, via its selective engagement of the membrane estrogen receptor GqMER. Using both neuronal culture assays and transgenic mouse models of Alzheimer’s disease (AD), we have shown that STX protects against the deleterious effects of β-amyloid (Aβ), in part by supporting mitochondrial function and synaptic integrity. However, the specific transduction pathways by which STX induces these beneficial responses have not been previously investigated.MethodsUsing the MC65 neuroblastoma model of Aβ toxicity and primary cultures of hippocampal neurons from the 5XFAD mouse model of AD, we analyzed the involvement of different signal transduction pathways associated with STX-dependent responses in other contexts. We used pharmacological methods to test the role of key pathway components in assays of cell viability, neuronal morphology, quantitative immunoblots to analyze pathway engagement, and modulation of the mitochondrial permeability transition pore.ResultsWe found that the neuroprotective effects of STX against Aβ toxicity required engagement of the PI3K/Akt/GSK3β pathway. Using well-characterized inhibitors of specific isoforms of the p110 catalytic domain of PI3K, we then showed that this response was predominantly mediated via engagement of the P110δ isoform, with a more modest contribution by P110β. In contrast, targeting the PLC/PKC/PKA pathway (which plays a prominent role in hypothalamic neurons) had a relatively modest effect on the neuroprotective responses induced by STX, while targeting ERK/MAPK signaling had no significant effect.DiscussionIn combination with our previous studies, these results indicate that engagement of GqMER by STX promotes neuroprotective responses via convergent signaling pathways that mitigate the effects of Aβ toxicity on mitochondrial function, synaptic integrity, and neuronal calcium (Ca2+) homeostasis. They also provide the framework for testing the mechanisms of STX neuroprotection in vivo, using mouse AD models. Since STX has been shown to provide many of the beneficial effects of 17β-estradiol in the brain without its adverse side effects (including feminizing effects in males), these results support the hypothesis that STX might have therapeutic potential in patients at risk of AD.

Mitochondrial dysfunction is increasingly recognized as a key driver of pathology in patients with Parkinson's disease (PD). Peripheral blood mononuclear cells (PBMCs) have emerged as an accessible way to characterize mitochondrial function in PD. The aim of this study was to conduct a preliminary evaluation of the clinical relevance of PBMC mitochondrial function as a biomarker in early PD. PBMC mitochondrial bioenergetics were measured using the Seahorse XF platform in individuals with de novo, untreated PD (n = 13) and compared to age- and sex-matched healthy controls (n = 15). Correlations between mitochondrial endpoints and clinical outcomes were assessed in the PD group. Basal and ATP-linked respiration were elevated in the PD group (p = 0.002, p = 0.004), while spare capacity, or the cell's ability to handle an unexpected energetic stress, was decreased (p = 0.045), relative to controls. Notably, the variability within basal respiratory measurements was markedly increased in the PD group compared to controls (p = 0.002). Further analyses revealed significant correlations between spare capacity and clinical motor function scores within the PD group. These findings support the potential of PBMC bioenergetics as a biomarker for early-stage PD as well as disease progression.

Neuroinflammation and mitochondrial dysfunction are early events in Alzheimer’s disease (AD) and contribute to neurodegeneration and cognitive impairment. Evidence suggests that the inflammatory axis mediated by macrophage migration inhibitory factor (MIF) binding to its receptor, CD74, plays an important role in many central nervous system (CNS) disorders such as AD. Our group has developed DRhQ, a novel CD74 binding construct which competitively inhibits MIF binding, blocks macrophage activation and migration into the CNS, enhances anti-inflammatory microglia cell numbers and reduces pro-inflammatory gene expression. Here, we evaluate its effects in amyloid-β (Aβ) overexpressing mice. 5xFAD mice and their wild type littermates were treated with DRhQ (100 µg) or vehicle for 4 weeks. DRhQ improved cognition and cortical mitochondrial function in both male and female 5xFAD mice. Aβ plaque burden in 5xFAD animals was not robustly impacted by DRhQ treatment in either the hippocampus or the cortex. Cortical microglial activation was similarly not apparently affected by DRhQ treatment, although in the hippocampus there was evidence of a reduction in activated microglia for female 5xFAD mice. Future studies are needed to confirm this possible sex-dependent response on microglial activation, as well as to optimize the dose and timing of DRhQ treatment and gain a better understanding of its mechanism of action in AD.

Background/Objectives: Extracts of the plant Centella asiatica can enhance mitochondrial function, promote antioxidant activity and improve cognitive deficits. Asiatic acid (AA) is one of the constituent triterpene compounds present in the plant. In this study, we explore the effects of AA on brain mitochondrial function, antioxidant response and cognition in a beta-amyloid (Aβ)-overexpressing 5xFAD mouse line. Methods: Six- to seven-month-old 5xFAD mice were treated with 1% AA for 4 weeks. In the last week of treatment, associative memory was assessed along with mitochondrial bioenergetics and the expression of mitochondrial and antioxidant response genes from isolated cortical synaptosomes. The Aβ plaque burden was also evaluated. Results: AA treatment resulted in improvements in associative memory in female 5xFAD mice without altering the Aβ plaque burden. Cortical mitochondrial function and mitochondrial gene expression were increased in the AA-treated female 5xFAD mice, as was the expression of antioxidant genes. More modest effects of AA on cortical mitochondrial function and mitochondrial and antioxidant gene expression were observed in male 5xFAD mice. Conclusions: Oral AA treatment improved cognitive and mitochondrial function and activated antioxidant in Aβ-overexpressing mice. These changes occurred independent of alterations in Aβ plaque burden, suggesting that AA could have translational therapeutic relevance in later-stage AD when plaques are well established.

Background/objectives: A water extract of the plant Centella asiatica (CAW) has been shown to improve cognitive deficits in aged mice when administered for 5 weeks in drinking water. However, the contribution of the constituent compounds within CAW to the beneficial effects of the extract remains unelucidated. This study evaluated the effects of triterpene (TT) and caffeoylquinic acids (CQA) found within CAW, on learning, cognitive flexibility, memory, and anxiety-like behaviors in aged C57BL6 mice. Methods: Eighteen-month-old male and female C57BL6 mice were administered either TT, CQA, or the combination (TT+CQA) in their drinking water for a total of 5 weeks, at concentrations corresponding to their presence in CAW. During the final two weeks of treatment learning, executive function, memory, and anxiety were assessed. Results: Aged mice of both sexes showed significant improvement in learning when treated with TT and CQA separately and in combination. Treatment with TT also improved cognitive flexibility in aged mice of both sexes, but CQA and the combination of TT+CQA did not alter cognitive flexibility in aged male mice. There was no effect on recognition memory or anxiety in any of the treatment groups (TT, CQA, TT+CQA) relative to mice treated with the vehicle control although there was a trend towards improved recognition memory with TT treatment. Conclusions: These results suggest that the TT and CQA present in CAW likely contribute to its previously reported amelioration of age-related cognitive changes, especially in learning and cognitive flexibility, while other constituents may be responsible for CAW’s anxiolytic effects.

Alterations in epigenetic modifications, like DNA methylation, in peripheral blood could serve as a useful, minimally invasive biomarker of the effects of anti-aging interventions. This study explores this potential with a water extract of the botanical Centella asiatica (CAW). Eighteen-month-old mice were treated with CAW in their drinking water for 5 weeks alongside vehicle-treated eighteen-month-old C57BL6 mice. Reduced representation bisulfite sequencing (RRBS) was used to identify genome-wide differential methylation in the blood of CAW-treated aged mice compared to vehicle-treated aged mice. Our results showed a distinct enrichment of differentially methylated regions (DMRs) nearby genes involved in biological processes relevant to aging (i.e., antioxidant response, metabolic regulation, cellular metabolism). A distinct difference was observed between males and females in both the number of methylation sites and the state of methylation. Moreover, genes nearby or overlapping DMRs were found to be enriched for biological processes related to previously described cellular effects of CAW in the mouse brain (i.e., antioxidant response, metabolic regulation, calcium regulation, and circadian rhythm). Together, our data suggest that the peripheral blood methylation signature of CAW in the blood could be a useful, and readily accessible, biomarker of CAW’s effects in aging.

Recent studies demonstrate that Parkinson’s disease (PD) is associated with dysregulated metabolic flux through the kynurenine pathway (KP), in which tryptophan is converted to kynurenine (KYN), and KYN is subsequently metabolized to neuroactive compounds quinolinic acid (QA) and kynurenic acid (KA). Here, we used mass-spectrometry to compare blood and cerebral spinal fluid (CSF) KP metabolites between 158 unimpaired older adults and 177 participants with PD. We found increased neuroexcitatory QA/KA ratio in both plasma and CSF of PD participants associated with peripheral and cerebral inflammation and vitamin B6 deficiency. Furthermore, increased QA tracked with CSF tau, CSF soluble TREM2 (sTREM2) and severity of both motor and non-motor PD clinical symptoms. Finally, PD patient subgroups with distinct KP profiles displayed distinct PD clinical features. These data validate the KP as a site of brain and periphery crosstalk, integrating B-vitamin status, inflammation and metabolism to ultimately influence PD clinical manifestation.

Centella asiatica (L.) Urban (Apiaceae) has been utilized for centuries in traditional medicine systems in Southeast Asia and Southern Africa, including Madagascar. Previous studies have reported evidence of the therapeutic potential of C. asiatica formulations in models of Alzheimer's Disease and other dementias. Caffeoylquinic acids (CQAs) have been identified to be among the pharmacologically relevant metabolites contributing to the botanical's cognitive enhancement and neuroprotective effects. Isomers of CQAs are, however, difficult to differentiate by commonly used LC-MS techniques, making the characterization, standardization, and batch-to-batch consistency of these formulations challenging. Individual CQAs have unique proton Spin Network Fingerprints (pSNFs) that can be used to distinguish between CQA regioisomers within complex extracts. This work describes the development of a CQA-focused pSNF library that can be used to complement LC-MS methods for the accurate metabolite identification and characterization of bioactive C. asiatica fractions and extracts. The isolation of two new (1 and 2) and four known (3-6) CQAs and CQA analogues from C. asiatica and their contribution to the pSNF library are also discussed herein.

Background: A water extract of the Ayurvedic plant Centella asiatica (L.) Urban, family Apiaceae (CAW), improves cognitive function in mouse models of aging and Alzheimer’s disease and affects dendritic arborization, mitochondrial activity, and oxidative stress in mouse primary neurons. Triterpenes (TT) and caffeoylquinic acids (CQA) are constituents associated with these bioactivities of CAW, although little is known about how interactions between these compounds contribute to the plant’s therapeutic benefit. Methods: Mouse primary cortical neurons were treated with CAW or equivalent concentrations of four TT combined, eight CQA combined, or these twelve compounds combined (TTCQA). Treatment effects on the cell transcriptome (18,491 genes) and metabolome (192 metabolites) relative to vehicle control were evaluated using RNAseq and metabolomic analyses, respectively. Results: Extensive differentially expressed genes (DEGs) were seen with all treatments, as well as evidence of interactions between compounds. Notably, many DEGs seen with TT treatment were not observed in the TTCQA condition, possibly suggesting CQA reduced the effects of TT. Moreover, additional gene activity seen with CAW as compared to TTCQA indicates the presence of additional compounds in CAW that further modulate TTCQA interactions. Weighted Gene Correlation Network Analysis (WGCNA) identified 4 gene co-expression modules altered by treatments that were associated with extracellular matrix organization, fatty acid metabolism, cellular response to stress and stimuli, and immune function. Compound interaction patterns were seen at the eigengene level in these modules. Interestingly, in metabolomics analysis, the TTCQA treatment saw the highest number of changes in individual metabolites (20), followed by CQA (15), then TT (8), and finally CAW (3). WGCNA analysis found two metabolomics modules with significant eigenmetabolite differences for TT and CQA and possible compound interactions at this level. Conclusions: Four gene expression modules and two metabolite modules were altered by the four treatment types applied. This methodology demonstrated the existence of both negative and positive interactions between TT, CQA, and additional compounds found in CAW on the transcriptome and metabolome of mouse primary cortical neurons.

ETHNOPHARMACOLOGICAL RELEVANCE:Withania somnifera (WS), also known as ashwagandha, is used in traditional Ayurvedic medicine for its rejuvenating properties. While most research has focused on withanolides as primary bioactive constituents, this study highlights the potential role of alkaloids in the biological effects of WS. AIM OF THE STUDY:To explore compounds responsible for bioactivity of WS on dendritic complexity in mouse primary neurons and on stress-related behaviors in Drosophila melanogaster. MATERIALS AND METHODS:An aqueous extract was prepared from dried root of WS grown in Central Oregon, and fractionated into ethanol insoluble, polar, and non-polar fractions. The preparations were tested for effects on dendritic complexity of mouse primary hippocampal neurons, and on stress-induced behavioral changes in Drosophila melanogaster flies. The composition of WS extract and fractions was examined using liquid chromatography coupled to high-resolution or multiple reaction monitoring mass spectrometry (LC-HRMS/MS or LC-MRM-MS). RESULTS:WS root aqueous extract and its polar fraction enhanced dendritic complexity in mouse primary hippocampal neurons and significantly improved stress-related behavior in Drosophila, whereas the withanolide-rich, non-polar fraction did not. The bioactive, polar fraction contained alkaloids including acetyltropine, which was then confirmed as a major, bioactive alkaloid in WS root and differentiated from its isomer acetyl exotropine by its LC-MRM-MS retention time and 2D-NMR. CONCLUSIONS:These findings highlight a role for alkaloids, including acetyltropine, as potential active constituents of WS associated with neuroprotective and anti-stress effects traditionally attributed to this plant. Results support continued evaluation of additional WS active compounds beyond withanolides.

Introduction:CSF α-klotho levels might affect Aβ40, Aβ42, and the Aβ42/40 ratio in the cerebrospinal fluid (CSF). Methods:CSF α-klotho was assayed in ovariectomized rhesus macaques (NHPs) maintained on a Western-style diet (WSD) to assess the effect of estrogen hormone therapy (HT). CSF and serum α-klotho was also analyzed in females and males of different ages and whether it was associated with Aβ42, Aβ40, or the Aβ42/40 ratio. Furthermore, CSF and serum α-klotho were analyzed in women and men with dementia and controls and whether they were associated with CSF Aβ levels. Results:HT was associated with increased CSF α-klotho levels. Furthermore, α-klotho and Aβ levels were correlated in a species- and cognitive health-dependent fashion. Higher CSF and serum levels of α-klotho were seen in controls than in patients with dementia. Discussion:Understanding the species differences in the beneficial effects of α-klotho on CSF Aβ physiology should open new avenues for treating AD.

Age-related sleep disruption is common in older adults. Not only does the total amount of time spent in sleep decline, but also the number of arousals during sleep increases with age. As sleep is important for both memory consolidation and to prevent neurodegenerative pathology, this decline in sleep and/or sleep consolidation may underlie age-related cognitive decline and dementias. Furthermore, treatment of sleep disruption can improve quality of life. However, few interventions have successfully reversed age-related sleep decline. Extracts from the plant Centella asiatica have demonstrated neuroprotective effects in human, rodent, and fly models of aging and neurodegenerative diseases and is a promising intervention for dementias, yet little is known about how these extracts affect sleep patterns. Here, we administered chow containing Centella asiatica water extract (CAW) or control chow to male and female C57BL6/J mice aged 18 months. Effects on sleep composition were determined using electrodes that recorded electroencephalogram (EEG) and electromyogram (EMG) signals. We found that chow containing CAW (1000 mg/kg/day) increased the duration of rapid eye movement (REM) sleep and theta power during REM sleep in aged male mice as well as decreased the number of arousals during sleep observed in aged females, compared to age- and sex-matched controls. We conclude that CAW administered in food has a moderate, sex-dependent effect on sleep quantity and quality.Statement of Significance Sleep declines with age and may underline age-related cognitive changes. However, few interventions have successfully reversed age-related sleep and cognitive decline. This study found that an extract from the plant Centella asiatica increased REM sleep duration in aged male mice and decreased sleep fragmentation in aged female mice, compared to age- and sex-matched controls. Whether these moderate, sex-dependent effect sizes on sleep in aged mice are impactful enough to affect cognition, quality of life, and/or neurodegenerative pathology could be explored in future studies.

Neuroinflammation and mitochondrial dysfunction are early events in Alzheimer’s disease (AD) and contribute to neurodegeneration and cognitive impairment. Evidence suggests that the inflammatory axis mediated by macrophage migration inhibitory factory (MIF) binding to its receptor, CD74, plays an important role in many central nervous system (CNS) disorders like AD. Our group has developed DRhQ, a novel CD74 binding construct that competitively inhibits MIF binding, blocks T-cell and macrophage activation and migration into the CNS, enhances anti-inflammatory microglia cell numbers and reduces pro-inflammatory gene expression. Here we evaluate its effects in β-amyloid (Aβ) overexpressing mice. 5xFAD mice and their wild type littermates were treated with DRhQ (100 µg) or vehicle for 4 weeks. DRhQ improved cognition and cortical mitochondrial function in both male and female 5xFAD mice. Aβ plaque burden in 5xFAD animals were not robustly impacted by DRhQ treatment nor was microglial activation, although in the hippocampus there was some evidence of a reduction in female 5xFAD mice. Future studies are needed to confirm this possible sex-dependent response on microglial activation as well as to optimize the dose, and timing of DRhQ treatment and gain a better understanding of its mechanism of action.### Competing Interest StatementThe authors have declared no competing interest.