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Lipids are critical to brain structure and function, accounting for approximately 50% of its dry weight. However, the impact of aging on brain lipids remains poorly characterized. To address this, here we applied three complementary mass spectrometry techniques: multiple reaction monitoring (MRM) profiling, untargeted liquid chromatography tandem mass spectrometry (LC-MS/MS), and desorption electrospray ionization-MS imaging (DESI-MSI). We used brains from mice of three age groups: adult (3-4 months), middle-aged (10 months), and old (19-21 months). Phospholipids such as phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol were more abundant, while phosphatidylinositol and phosphatidylserine were reduced in old mice compared to adults or middle-aged mice. Key lipids such as polyunsaturated fatty acids, including DHA, AA, HexCer, SHexCer, and SM, were among the most abundant lipids in aged brains. DESI-MSI revealed spatial lipid distribution patterns consistent with findings from MRM profiling and LC-MS/MS. Integration of lipidomic data with the recently published proteomics data from the same tissues highlighted changes in proteins and phosphorylation levels of several proteins associated with Cer, HexCer, FA, PI, SM, and SHexCer metabolism, aligning with the multiplatform lipid surveillance. These findings shed insight into age-dependent brain lipid changes and their potential contribution to age-related cognitive decline.

A central hallmark of neurodegenerative diseases is the irreversible accumulation of misfolded proteins in the brain by aberrant phosphorylation. Understanding the mechanisms underlying protein phosphorylation and its role in pathological protein aggregation within the context of aging is crucial for developing therapeutic strategies aimed at preventing or reversing such diseases. Here, we applied multi-protease digestion and quantitative mass spectrometry to compare and characterize dysregulated proteins and phosphosites in the mouse brain proteome using three different age groups: young-adult (3-4 months), middle-age (10 months), and old mice (19-21 months). Proteins associated with senescence, neurodegeneration, inflammation, cell cycle regulation, the p53 hallmark pathway, and cytokine signaling showed significant age-dependent changes in abundances and level of phosphorylation. Several proteins implicated in Alzheimer's disease (AD) and Parkinson's disease (PD) including tau (Mapt), Nefh, and Dpysl2 (also known as Crmp2) were hyperphosphorylated in old mice brain suggesting their susceptibility to the diseases. Cdk5 and Gsk3b, which are known to phosphorylate Dpysl2 at multiple specific sites, had also increased phosphorylation levels in old mice suggesting a potential crosstalk between them to contribute to AD. Hapln2, which promotes α-synuclein aggregation in patients with PD, was one of the proteins with highest abundance in old mice. CD9, which regulates senescence through the PI3K-AKT-mTOR-p53 signaling was upregulated in old mice and its regulation was correlated with the activation of phosphorylated AKT1. Overall, the findings identify a significant association between aging and the dysregulation of proteins involved in various pathways linked to neurodegenerative diseases with potential therapeutic implications., Competing Interests: Conflict of interest The authors declare no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The appearance of misfolded and aggregated proteins is a pathological hallmark of numerous neurodegenerative diseases including Alzheimer's disease and Parkinson's disease. Sleep disruption is proposed to contribute to these pathological processes and is a common early feature among neurodegenerative disorders. Synucleinopathies are a subclass of neurodegenerative conditions defined by the presence of α-synuclein aggregates, which may not only enhance cell death, but also contribute to disease progression by seeding the formation of additional aggregates in neighboring cells. The mechanisms driving intercellular transmission of aggregates remains unclear. We propose that disruption of sleep-active glymphatic function, caused by loss of precise perivascular AQP4 localization, inhibits α-synuclein clearance and facilitates α-synuclein propagation and seeding. We examined human post-mortem frontal cortex and found that neocortical α-synuclein pathology was associated with AQP4 mis-localization throughout the gray matter. Using a transgenic mouse model lacking the adapter protein α-syntrophin, we observed that loss of perivascular AQP4 localization impairs the glymphatic clearance of α-synuclein from intersititial to cerebrospinal fluid. Using a mouse model of α-synuclein propogation, using pre-formed fibril injection, we observed that loss of perivascular AQP4 localization increased α-synuclein aggregates. Our results indicate α-synuclein clearance and propagation are mediated by glymphatic function and that AQP4 mis-localization observed in the presence of human synucleinopathy may contribute to the development and propagation of Lewy body pathology in conditions such as Lewy Body Dementia and Parkinson's disease.

In this article, we propose a post-processing scheme for the novel volumetric microscopy technique SILMAS. We demonstrate this scheme on data from an alpha-synuclein transgenic mouse brain. By combining structured illumination and axial sweeping, a SILMAS measurement provides a prerequisite for quantitative data extraction through improved contrast and optical sectioning. However, due to the technique's efficient removal ofb multiple scattered light, image artifacts such as illumination inhomogeneity, shadowing stripes, and signal attenuation, are highlighted in the recorded volumes. To suppress these artifacts, we rely on the strengths of the imaging method. The SILMAS data, together with the Beer-Lambert law, allow for an approximation of real light extinction, which can be used to compensate for light attenuation in a near-quantitative way. Shadowing stripes can be suppressed efficiently using a computational strategy thanks to the large numerical aperture of an axially swept light sheet. Here, we build upon prior research that employed wavelet-Fourier filtering by incorporating an extra bandpass step. This allows us to filter high-contrast light sheet microscopy data without introducing new artifacts and with minimal distortion of the data. The combined technique is suitable for imaging cleared tissue samples of up to a centimeter scale with an isotropic resolution of a few microns. The combination of a thin and uniform light sheet, scattered light suppression, light attenuation compensation, and shadowing suppression produces volumetric data that is seamless and highly uniform., Competing Interests: The authors declare no conflicts of interest., (© 2024 The Author(s).)

Light-sheet fluorescent microscopy (LSFM) has, in recent years, allowed for rapid 3D-imaging of cleared biomedical samples at larger and larger scale. However, even in cleared samples, multiple light scattering often degrades the imaging contrast and widens the optical sectioning. Accumulation of scattering intensifies these negative effects as light propagates inside the tissue, which accentuates the issues when imaging large samples. With axially swept light-sheet microscopy (ASLM), centimeter-scale samples can be scanned with a uniform micrometric optical sectioning. But to fully utilize these benefits for 3D-imaging in biomedical tissue samples, suppression of scattered light is needed. Here, we address this by merging ASLM with light-sheet based structured illumination into Structured Illumination Light-sheet Microscopy with Axial Sweeping (SILMAS). The SILMAS method thus enables high-contrast imaging, isotropic micrometric resolution and uniform optical sectioning in centimeter-scale scattering samples, creating isotropic 3D-volumes of e.g., whole mouse brains without the need for any computation-heavy post-processing. We demonstrate the effectiveness of the approach in agarose gel phantoms with fluorescent beads, and in an PFF injected alpha-synuclein transgenic mouse model tagged with a green fluorescent protein (SynGFP). SILMAS imaging is compared to standard ASLM imaging on the same samples and using the same optical setup, and is shown to increase contrast by as much as 370% and reduce widening of optical sectioning by 74%. With these results, we show that SILMAS improves upon the performance of current state-of-the-art light-sheet microscopes for large and imperfectly cleared tissue samples and is a valuable addition to the LSFM family., Competing Interests: The authors declare no conflicts of interest., (© 2022 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement.)

It is necessary to develop an understanding of the specific mechanisms involved in alpha-synuclein aggregation and propagation to develop disease modifying therapies for age-related synucleinopathies, including Parkinson's disease and Dementia with Lewy Bodies. To adequately address this question, we developed a new transgenic mouse model of synucleinopathy that expresses human A53T SynGFP under control of the mouse prion protein promoter. Our characterization of this mouse line demonstrates that it exhibits several distinct advantages over other, currently available, mouse models. This new model allows rigorous study of the initial location of Lewy pathology formation and propagation in the living brain, and strongly suggests that aggregation begins in axonal structures with retrograde propagation to the cell body. This model also shows expeditious development of alpha-synuclein pathology following induction with small, in vitro-generated alpha-synuclein pre-formed fibrils (PFFs), as well as accelerated cell death of inclusion-bearing cells. Using this model, we found that aggregated alpha-synuclein somatic inclusions developed first in neurons, but later showed a second wave of inclusion formation in astrocytes. Interestingly, astrocytes appear to survive much longer after inclusion formation than their neuronal counterparts. This model also allowed careful study of peripheral-to-central spread of Lewy pathology after PFF injection into the hind limb musculature. Our results clearly show evidence of progressive, retrograde trans-synaptic spread of Lewy pathology through known neuroanatomically connected pathways in the motor system. As such, we have developed a promising tool to understand the biology of neurodegeneration associated with alpha-synuclein aggregation and to discover new treatments capable of altering the neurodegenerative disease course of synucleinopathies.

Alpha-synuclein is a presynaptic protein that forms abnormal cytoplasmic aggregates in Lewy body disorders. Although nuclear alpha-synuclein localization has been described, its function in the nucleus is not well understood. We demonstrate that alpha-synuclein modulates DNA repair. First, alpha-synuclein colocalizes with DNA damage response components within discrete foci in human cells and mouse brain. Removal of alpha-synuclein in human cells leads to increased DNA double-strand break (DSB) levels after bleomycin treatment and a reduced ability to repair these DSBs. Similarly, alpha-synuclein knock-out mice show increased neuronal DSBs that can be rescued by transgenic reintroduction of human alpha-synuclein. Alpha-synuclein binds double-stranded DNA and helps to facilitate the non-homologous end-joining reaction. Using a new, in vivo imaging approach that we developed, we find that serine-129-phosphorylated alpha-synuclein is rapidly recruited to DNA damage sites in living mouse cortex. We find that Lewy inclusion-containing neurons in both mouse model and human-derived patient tissue demonstrate increased DSB levels. Based on these data, we propose a model whereby cytoplasmic aggregation of alpha-synuclein reduces its nuclear levels, increases DSBs, and may contribute to programmed cell death via nuclear loss-of-function. This model could inform development of new treatments for Lewy body disorders by targeting alpha-synuclein-mediated DNA repair mechanisms.

Voice and swallowing deficits can occur with aging. Tongue exercise paired with a swallow may be used to treat swallowing disorders, but may also benefit vocal function due to cross-system activation effects. It is unknown how exercise-based neuroplasticity contributes to behavior and maintenance following treatment. Eighty rats were used to examine behavioral parameters and changes in neurotrophins after tongue exercise paired with a swallow. Tongue forces and ultrasonic vocalizations were recorded before and after training/detraining in young and old rats. Tissue was analyzed for neurotrophin content. Results showed tongue exercise paired with a swallow was associated with increased tongue forces at all ages. Gains diminished after detraining in old rats. Age-related changes in vocalizations, neurotrophin 4 (NT4), and brain derived neurotrophic factor (BDNF) were found. Minimal cross-system activation effects were observed. Neuroplastic benefits were demonstrated with exercise in old rats through behavioral improvements and up-regulation of BDNF in the hypoglossal nucleus. Tongue exercise paired with a swallow should be developed, studied, and optimized in human clinical research to treat swallowing and voice disorders in elderly people., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Unilateral lesions to the medial forebrain bundle with 6-hydroxydopamine (6-OHDA) lead to force and timing deficits during a complex licking task. We hypothesized that training targeting tongue force generation during licking would improve timing and force measures and also lead to striatal dopamine sparing. Nine month-old male Fisher344/Brown Norway rats were used in this experiment. Sixteen rats were in the control condition and received tongue exercise (n=8) or no exercise (n=8). Fourteen rats were in the 6-OHDA lesion condition and underwent tongue exercise (n=7) and or no exercise (n=7). Following 4 weeks of training and post-training measures, all animals underwent bilateral stimulation of the hypoglossal nerves to measure muscle contractile properties and were then transcardially perfused and brain tissues collected for immunohistochemistry to examine striatal dopamine content. Results demonstrated that exercise animals performed better for maximal force, average force, and press rate than their no-exercise counterparts, and the 6-OHDA animals that underwent exercise performed as well as the Control No Exercise group. Interestingly, there were no group differences for tetanic muscle force, despite behavioral recovery of forces. Additionally, behavioral and neurochemical analyses indicate that there were no differences in striatal dopamine. Thus, targeted exercise can improve tongue force and timing deficits related to 6-OHDA lesions and this exercise likely has a central, versus peripheral (muscle strength) mechanism. However, this mechanism is not related to sparing of striatal dopamine content., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Introduction: Age-related decreases in tongue muscle mass and strength have been reported. It may be possible to prevent age-related tongue muscle changes using neuromuscular electrical stimulation (NMES). Our hypothesis was that alterations in muscle contractile properties and myosin heavy chain composition would be found after NMES., Methods: Fifty-four young, middle-aged, and old 344/Brown Norway rats were included in this study. Twenty-four rats underwent bilateral electrical stimulation of the hypoglossal nerves for 8 weeks and were compared with control or sham rats. Muscle contractile properties and myosin heavy chain (MHC) in the genioglossus (GG), styloglossus (SG), and hyoglossus (HG) muscles were examined., Results: Compared with unstimulated control rats, we found reduced muscle fatigue, increased contraction and half-decay times, and increased twitch and tetanic tension. Increased type I MHC was found, except for in GG in old and middle-aged rats., Conclusion: Transitions in tongue muscle contractile properties and phenotype were found after NMES., (Copyright © 2012 Wiley Periodicals, Inc.)

Age-associated changes in tongue musculature may contribute to dysphagia. One possible treatment is tongue exercise. Exercise induces synaptic plasticity by increasing neurotrophic factors in spinal cord and limb musculature. However, effects of exercise on neurotrophic factors in the cranial sensorimotor system are unknown. Our purpose was to examine the effects of age and exercise on brain-derived neurotrophic factor (BDNF) and its receptor TrkB in the rat hypoglossal nucleus. Young, middle-aged, and old rats were assigned to exercise or no-exercise control conditions. Exercise animals were trained to perform a tongue press task for 8 weeks. Samples from the hypoglossal nucleus were analyzed for BDNF and TrkB immunoreactivity. Baseline maximum tongue forces were similar in all age groups and increased significantly following exercise. BDNF immunoreactivity did not show a significant decrease with age in control group. However, in the exercise group, BDNF was significantly increased in young animals. TrkB immunoreactivity decreased significantly with age in control group, but did not change with exercise. BDNF and TrkB immunoreactivity levels were positively correlated with exercise in young and middle aged animals, but were negatively or weakly correlated with exercise in old animals and with a lack of exercise in no-exercise controls. Tongue exercise was associated with increased tongue forces in rats at all ages. While increases in BDNF and TrkB levels associated with exercise may play a role in mechanisms contributing to increased tongue forces in young and middle-aged rats, other mechanisms may be involved in increased tongue forces observed in old rats., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Deficits in tongue function in conjunction with airway compromise can contribute to dysphagia associated with Parkinson disease (PD). However, it is unknown if these deficits are related to the primary disease pathology in PD, nigrostriatal dopamine depletion. To directly study the impact of striatal dopamine depletion on tongue function, we used unilateral infusion of the neurotoxin 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle and measured tongue force and timing parameters during a complex tongue protrusion task for a water reward. Maximal and average forces were significantly diminished and average press time was significantly longer after neurotoxin administration, reflecting aspects of bradykinesia and hypokinesia associated with PD. Our findings suggest that even unilateral deficits to the nigrostriatal dopamine system may be contributing to some of the lingual sensorimotor deficits seen in PD. Because previous research in rat models of PD has shown that targeted training of the limb can rescue behavioral deficits and spare striatal dopamine neurons, early intervention for cranial sensorimotor deficits may also be indicated., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Age-related tongue weakness may contribute to swallowing deficits in the elderly. One contributing factor may be an alteration in muscle-fiber-type properties with aging. However, it is not clear how muscle fiber types within the aged tongue may vary from those found in young adults, or how fiber types may vary across the anteroposterior axis of the extrinsic tongue muscles. We examined the myosin heavy chain (MHC) composition of anterior, medial, and posterior sections of the genioglossus muscle (GG) in ten old male Fischer 344/Brown Norway rats and compared findings to previously reported data from young adult male rats. Significant differences (p < 0.01) between young adult and old rats were found in the distribution of MHC isoforms along the anteroposterior axis of the muscle. In the anterior, medial, and posterior regions, there was a significantly smaller proportion of type IIb MHC in the old rat GG muscles, while the proportion of type IIx MHC was significantly greater. In the medial region, the proportion of type I MHC was found to be significantly greater in the old rats. Thus, we found a shift to more slowly contracting muscle fibers in the aged rat tongue.