Accumulation of alkyl-lysophosphatidylcholines in Niemann-Pick disease type C1.

Lysosomal function is impaired in Niemann-Pick disease type C1 (NPC1), a rare and inherited neurodegenerative disorder, resulting in late endosomal/lysosomal accumulation of unesterified cholesterol. The precise pathogenic mechanism of NPC1 remains incompletely understood. In this study, we employed metabolomics to uncover secondary accumulated substances in NPC1. Our findings unveiled a substantial elevation in the levels of three alkyl-lysophosphatidylcholine [alkyl-LPC, also known as lyso-platelet activating factor (PAF)] species in NPC1 compared to controls across various tissues, including brain tissue from individuals with NPC1, liver, spleen, cerebrum, cerebellum, and brain stem from NPC1 mice, as well as in both brain and liver tissue from NPC1 cats. The three elevated alkyl-LPC species were as follows: LPC O-16:0, LPC O-18:1, and LPC O-18:0. However, the levels of PAF 16:0, PAF 18:1, and PAF 18:0 were not altered in NPC1. In the NPC1 feline model, the brain and liver alkyl-LPC levels were reduced following 2-hydroxypropyl-ß-cyclodextrin (HPßCD) treatment, suggesting that alkyl-LPCs are secondary storage metabolites in NPC1 disease. Unexpectedly, cerebrospinal fluid (CSF) levels of LPC O-16:0 and LPC O-18:1 were decreased in individuals with NPC1 compared to age-appropriate comparison samples, and their levels were increased in 80% of participants 2 years after intrathecal HPßCD treatment. The fold increases in CSF LPC O-16:0 and LPC O-18:1 levels were more pronounced in responders compared to nonresponders. This study identified alkyl-LPC species as secondary storage metabolites in NPC1 and indicates that LPC O-16:0 and LPC O-18:1, in particular, could serve as potential biomarkers for tracking treatment response in NPC1 patients.

Endogenous Protein-Protein Interaction Network of the NPC Cholesterol Transporter 1 in the Cerebral Cortex.

NPC intracellular cholesterol transporter 1 (NPC1) is a multipass, transmembrane glycoprotein mostly recognized for its key role in facilitating cholesterol efflux. Mutations in the NPC1 gene result in Niemann-Pick disease, type C (NPC), a fatal, lysosomal storage disease. Due to the progressively expanding implications of NPC1-related disorders, we investigated endogenous NPC1 protein-protein interactions in the mouse cortex and human-derived iPSCs neuronal models of the disease through coimmunoprecipitation-coupled with LC-MS based proteomics. The current study investigated protein-protein interactions specific to the wild-type and the most prevalent NPC1 mutation (NPC1I1061T) while filtering out any protein interactor identified in the Npc1-/- mouse model. Additionally, the results were matched across the two species to map the parallel interactome of wild-type and mutant NPC1I1061T. Most of the identified wild-type NPC1 interactors were related to cytoskeleton organization, synaptic vesicle activity, and translation. We found many putative NPC1 interactors not previously reported, including two SCAR/WAVE complex proteins that regulate ARP 2/3 complex actin nucleation and multiple membrane proteins important for neuronal activity at synapse. Moreover, we identified proteins important in trafficking specific to wild-type and mutant NPC1I1061T. Together, the findings are essential for a comprehensive understanding of NPC1 biological functions in addition to its classical role in sterol efflux.

Proteomics insights into fragile X syndrome: Unraveling molecular mechanisms and therapeutic avenues.

Fragile X Syndrome (FXS) is a neurodevelopment disorder characterized by cognitive impairment, behavioral challenges, and synaptic abnormalities, with a genetic basis linked to a mutation in the FMR1 (Fragile X Messenger Ribonucleoprotein 1) gene that results in a deficiency or absence of its protein product, Fragile X Messenger Ribonucleoprotein (FMRP). In recent years, mass spectrometry (MS) - based proteomics has emerged as a powerful tool to uncover the complex molecular landscape underlying FXS. This review provides a comprehensive overview of the proteomics studies focused on FXS, summarizing key findings with an emphasis on dysregulated proteins associated with FXS. These proteins span a wide range of cellular functions including, but not limited to, synaptic plasticity, RNA translation, and mitochondrial function. The work conducted in these proteomic studies provides a more holistic understanding to the molecular pathways involved in FXS and considerably enhances our knowledge into the synaptic dysfunction seen in FXS.

Follicular fluid aids cell adhesion, spreading in an age independent manner and shows an age-dependent effect on DNA damage in fallopian tube epithelial cells.

Ovarian cancer (OC) is deadly, and likely arises from the fallopian tube epithelium (FTE). Despite the association of OC with ovulation, OC typically presents in post-menopausal women who are no longer ovulating. The goal of this study was to understand how ovulation and aging interact to impact OC progression from the FTE. Follicular fluid released during ovulation induces DNA damage in the FTE, however, the role of aging on FTE exposure to follicular fluid is unexplored. Follicular fluid samples were collected from 14 women and its effects on FTE cells was assessed. Follicular fluid caused DNA damage and lipid oxidation in an age-dependent manner, but instead induced cell proliferation in a dose-dependent manner, independent of age in FTE cells. Follicular fluid regardless of age disrupted FTE spheroid formation and stimulated attachment and growth on ultra-low attachment plates. Proteomics analysis of the adhesion proteins in the follicular fluid samples identified vitronectin, a glycoprotein responsible for FTE cell attachment and spreading.

In Vivo Simvastatin and Brain Radiation in a Model of HER2 + Inflammatory Breast Cancer Brain Metastasis.

Purpose: Inhibiting HMG-CoA reductase with simvastatin prevents breast cancer metastases in preclinical models and radiosensitizes monolayer and stem-like IBC cell lines in vitro . Given the extensive use of simvastatin worldwide and its expected penetration into the brain, we examined whether regulating cholesterol with simvastatin affected IBC3 HER2+ brain metastases. Methods and Materials: Breast cancer cell lines KPL4 and MDA-IBC3 were examined in vitro for DNA repair after radiation with or without statin treatment. Brain metastasis endpoints were examined in the MDA-IBC3 brain metastasis model after ex vivo exposure to lipoproteins and after tail vein injections with and without whole-brain radiotherapy (WBR) and oral statin exposure. Results: Ex vivo preculture of MDA-IBC3 cells with very low-density lipoprotein (vLDL) enhanced the growth of colonized lesions in the brain in vivo compared with control or high-density lipoprotein (HDL), and concurrent oral simvastatin/ WBR reduced the incidence of micrometastatic lesions evaluated 10 days after WBR. However, statin, with or without WBR, did not reduce the incidence, burden, or number of macrometastatic brain lesions evaluated 5 weeks after WBR. Conclusions: Although a role for cholesterol biosynthesis is demonstrated in DNA repair and response to whole brain radiation in this model, durable in vivo efficacy of concurrent whole brain irradiation and oral statin was not demonstrated.

Gel-assisted mass spectrometry imaging enables sub-micrometer spatial lipidomics.

A technique capable of label-free detection, mass spectrometry imaging (MSI) is a powerful tool for spatial investigation of native biomolecules in intact specimens. However, MSI has often been precluded from single-cell applications due to the spatial resolution limit set forth by the physical and instrumental constraints of the method. By taking advantage of the reversible interaction between the analytes and a superabsorbent hydrogel, we have developed a sample preparation and imaging workflow named Gel-Assisted Mass Spectrometry Imaging (GAMSI) to overcome the spatial resolution limits of modern mass spectrometers. With GAMSI, we show that the spatial resolution of MALDI-MSI can be enhanced ~3-6-fold to the sub-micrometer level without changing the existing mass spectrometry hardware or analysis pipeline. This approach will vastly enhance the accessibility of MSI-based spatial analysis at the cellular scale.

Akap5 links synaptic dysfunction to neuroinflammatory signaling in a mouse model of infantile neuronal ceroid lipofuscinosis.

Palmitoylation and depalmitoylation represent dichotomic processes by which a labile posttranslational lipid modification regulates protein trafficking and degradation. The depalmitoylating enzyme, palmitoyl-protein thioesterase 1 (PPT1), is associated with the devastating pediatric neurodegenerative condition, infantile neuronal ceroid lipofuscinosis (CLN1). CLN1 is characterized by the accumulation of autofluorescent lysosomal storage material (AFSM) in neurons and robust neuroinflammation. Converging lines of evidence suggest that in addition to cellular waste accumulation, the symptomology of CLN1 corresponds with disruption of synaptic processes. Indeed, loss of Ppt1 function in cortical neurons dysregulates the synaptic incorporation of the GluA1 AMPA receptor (AMPAR) subunit during a type of synaptic plasticity called synaptic scaling. However, the mechanisms causing this aberration are unknown. Here, we used the Ppt1-/- mouse model (both sexes) to further investigate how Ppt1 regulates synaptic plasticity and how its disruption affects downstream signaling pathways. To this end, we performed a palmitoyl-proteomic screen, which provoked the discovery that Akap5 is excessively palmitoylated at Ppt1-/- synapses. Extending our previous data, in vivo induction of synaptic scaling, which is regulated by Akap5, caused an excessive upregulation of GluA1 in Ppt1-/- mice. This synaptic change was associated with exacerbated disease pathology. Furthermore, the Akap5- and inflammation-associated transcriptional regulator, nuclear factor of activated T cells (NFAT), was sensitized in Ppt1-/- cortical neurons. Suppressing the upstream regulator of NFAT activation, calcineurin, with the FDA-approved therapeutic FK506 (Tacrolimus) modestly improved neuroinflammation in Ppt1-/- mice. These findings indicate that the absence of depalmitoylation stifles synaptic protein trafficking and contributes to neuroinflammation via an Akap5-associated mechanism.

Optimization of systemic AAV9 gene therapy in Niemann-Pick disease type C1 mice.

Niemann-Pick disease, type C1 (NPC1) is a rare, fatal neurodegenerative disorder caused by pathological variants in NPC1, which encodes a lysosomal cholesterol transport protein. There are no FDA approved treatments for this disorder. Both systemic and central nervous system delivery of AAV9-hNPC1 have shown significant disease amelioration in NPC1 murine models. To assess the impact of dose and window of therapeutic efficacy in Npc1 m1N mice, we systemically administered three different doses of AAV9-hNPC1 at 4 weeks old and the medium dose at pre-, early, and post-symptomatic timepoints. Higher vector doses and treatment earlier in life were associated with enhanced transduction in the nervous system and resulted in significantly increased lifespan. Similar beneficial effects were noted after gene therapy in Npc1 I1061T mice, a model that recapitulates a common human hypomorphic variant. Our findings help define dose ranges, treatment ages, and efficacy in severe and hypomorphic models of NPC1 deficiency and suggest that earlier delivery of AAV9-hNPC1 in a pre-symptomatic disease state is likely to yield optimal outcomes in individuals with NPC1.

Temporal Alterations in White Matter in An App Knock-In Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is the most common form of dementia and results in neurodegeneration and cognitive impairment. White matter (WM) is affected in AD and has implications for neural circuitry and cognitive function. The trajectory of these changes across age, however, is still not well understood, especially at earlier stages in life. To address this, we used the AppNL-G-F/NL-G-F knock-in (APPKI) mouse model that harbors a single copy knock-in of the human amyloid precursor protein (APP) gene with three familial AD mutations. We performed in vivo diffusion tensor imaging (DTI) to study how the structural properties of the brain change across age in the context of AD. In late age APPKI mice, we observed reduced fractional anisotropy (FA), a proxy of WM integrity, in multiple brain regions, including the hippocampus, anterior commissure (AC), neocortex, and hypothalamus. At the cellular level, we observed greater numbers of oligodendrocytes in middle age (prior to observations in DTI) in both the AC, a major interhemispheric WM tract, and the hippocampus, which is involved in memory and heavily affected in AD, prior to observations in DTI. Proteomics analysis of the hippocampus also revealed altered expression of oligodendrocyte-related proteins with age and in APPKI mice. Together, these results help to improve our understanding of the development of AD pathology with age, and imply that middle age may be an important temporal window for potential therapeutic intervention.