Remyelination Improves Voiding Dysfunction in a Mouse Model of Multiple Sclerosis

Abstract Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS). Of note, over 80% of MS patients have urinary symptoms as one of their earliest symptoms. Since MS patients often live into older age, urinary incontinence and retention are significant problems affecting their quality of life. Although several studies show that inflammatory-demyelinating animal models of MS develop bladder dysfunction, the confounding influence of systemic inflammation in these models limits potential interpretation on the contribution of CNS-myelination to bladder dysfunction. We sought to address this knowledge gap using the cuprizone model of demyelination and remyelination. C57Bl/6 mice were treated with dietary cuprizone (0.2%w/w) for four weeks to induce demyelination. One group was allowed four additional weeks for recovery and remyelination. We performed voiding spot assay (VSA), urethane-anesthetized cystometry, and CNS-histology to assess demyelination-induced differences in urinary performance. We observed that cortical demyelination causes significant aberrance in voiding behavior (conscious cortical control) characterized by increased micturition frequency and reduced volume per micturition. Interestingly, remyelination restored healthy bladder function. However, there were no significant changes in the cystometric parameters (brainstem reflex) between the treatment groups. While MS is not classically considered a disease of aging, extending the longevity of these patients has not been reciprocated with improved treatments for their most-bothersome conditions, notably urinary symptoms that persist throughout life. Our data represent a novel compelling connection and strong correlation between CNS-myelination and cortical control of bladder function, which has potential implications in MS, aging, and aging-associated neurological disorders.

United States,3. Yale University,Yale University,Connecticut,United States Tau pathology emerges in a distinct spatial and temporal pattern in Alzheimer's Disease (AD).Anatomical studies in AD subjects and rhesus macaques show earliest signs of tau pathology in the stellate cell islands in entorhinal cortex (ERC) layer II.However, the molecular mechanisms that confer vulnerability to ERC layer II cells early in AD is unknown.cAMP-PKA magnification of calcium release has been seen in prefrontal cortex, associated with HCN channel opening to dynamically regulate synaptic strength.This process is regulated by phosphodiesterases (PDE), regulation that is lost with age.The current study examined whether this "signature of flexibility" could also be seen in layer II ERC, underlying vulnerability to tau pathology with aging.We used high-spatial resolution immunoEM to localize PDE4D and HCN1 in young rhesus macaque (7-10y) ERC layer II.Our results suggest that PDE4D was concentrated on the SER-spine apparatus and in postsynaptic density, and HCN1 expressed in the membrane near excitatory synapses in dendritic spines.Within dendritic shafts, PDE4D labeling was observed along microtubules and near mitochondria, whereas HCN1 was organized in discrete clusters along the plasma membrane.These data suggest that PDE4D is optimally positioned to modulate cAMP microdomains and control calcium extrusion from the SER.HCN1 channels are localized in subcompartments to facilitate dynamic physiological representation of sensory experience and visual space governed by cAMP-PKA signaling.The anatomical patterns in ERC layer II corroborate our findings in vulnerable glutamatergic circuits in prefrontal cortex, suggesting conserved molecular features in association cortices most susceptible in AD.

PHENOTHIAZINES TO TREAT ALZHEIMER'S DISEASE
Rachel Litke, 1 Bik Tzu Huang, 2 Damian Gonzalez, 2 Martine Rampanana, 2 Nicholas Grimaldi, 2 and Charles Mobbs, 3 1.icahn school of medicine at mount sinai, New York,New York,United States,2. Icahn School of Medicine at Mount Sinai,New York,New York,United States,3. Icahn School of Medicine at Mount Sinai,new york,New York,United States Current treatments of Alzheimer's Disease (AD) are largely ineffective and do not address underlying pathophysiological processes.The model organism C. elegans has been successfully used to discover compounds to treat human diseases, some now in clinical trials.To develop novel drugs and explore pathways to treat AD, we took on a forward pharmacological approach with a C. elegans model for AD, completed with studies to expand results to lifespan as well as healthspan.We screened 2560 drugs from the Microsource Spectrum library for their ability to delay proteotoxicity (indicated by paralysis) in an Abeta transgenic C. elegans muscle model of AD (CL2006) in liquid medium.Among the most protective drugs were phenothiazines, which are orally active and cross the blood-brain barrier, desirable properties of drugs to treat AD. 80 phenothiazines congeners were further assessed; 60% were protective in CL2006 worms.9/20 tested phenothiazines increased lifespan in N2 worms and 2/3 phenothiazines tested promoted significantly higher pharyngeal pumping rates compared with control till day 10 of adulthood in N2 worms. 2 of the drugs were protective in the C. elegans neuronal model of AD.This phenotypic screening approach led to the discovery of potential drugs to treat AD.These phenothiazines protect against Abeta toxicity, and assessment of efficacy to protect against other forms of proteotoxicity are ongoing.These studies suggest the utility of C. elegans to discover drugs to treat human diseases.Future studies will assess molecular mechanisms mediating the protective effects of these compounds.

QUANTIFICATION OF SIMILAR NEURODEGENERATION ACROSS GERIATRIC CONCUSSIONS AND ALZHEIMER'S DISEASE Andrei Irimia, and Kenneth Rostowsky, University of Southern California, University of Southern California, California, United States
Geriatric mild traumatic brain injury (mTBI) is a risk factor for Alzheimer's disease (AD), but few studies have studied how the neuroanatomic effects of these conditions can converge onto similar brain structure trajectories.Here we use magnetic resonance imaging to investigate similarities between mTBI and AD across both white and gray matter (WM and GM, respectively) using measures like fractional anisotropy (FA, a surrogate measure of WM integrity) and cortical thickness.We identify statistically significant similarities in neurodegeneration across mTBI (N = 33; age µ = 63 years (y), σ = 11 y) and AD (N = 66; age µ = 76 y, σ = 9 y) by testing for statistical equivalences of mean FA and cortical thickness.Both WM and GM are found to exhibit significant similarities in how mean FA and cortical thickness decrease, respectively, across mTBI and AD.For WM, the broadest spatial extent of statistical similarity between conditions, quantified as percentages of structures' volumes, is found within the superior fronto-occipital fasciculus (left (L): 91%, p < 0.05; right (R): 95%, p < 0.05), and in the crura of the fornix (L: 65%, p < 0.05; R: 80%, p < 0.05).Across mTBI and AD, cortical thinning trajectories are most similar in the superior precentral sulcus (L: 91%, p < 0.05; R: 100%, p < 0.05), and anterior lateral sulcus (L: 75%, p < 0.05; R: 86%, p < 0.05).Future studies should leverage such findings to identify AD risk factors in mTBI patients.

REMYELINATION IMPROVES VOIDING DYSFUNCTION IN A MOUSE MODEL OF MULTIPLE SCLEROSIS
Ramalakshmi Ramasamy, Cara Hardy, Stephen Crocker, and Phillip Smith, UConn Health, Farmington, Connecticut, United States Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS).Of note, over 80% of MS patients have urinary symptoms as one of their earliest symptoms.Since MS patients often live into older age, urinary incontinence and retention are significant problems affecting their quality of life.Although several studies show that inflammatory-demyelinating animal models of MS develop bladder dysfunction, the confounding influence of systemic inflammation in these models limits potential interpretation on the contribution of CNSmyelination to bladder dysfunction.We sought to address this knowledge gap using the cuprizone model of demyelination and remyelination.C57Bl/6 mice were treated with dietary cuprizone (0.2%w/w) for four weeks to induce demyelination.One group was allowed four additional weeks for recovery and remyelination.We performed voiding spot assay (VSA), urethane-anesthetized cystometry, and CNS-histology to assess demyelination-induced differences in urinary performance.We observed that cortical demyelination causes significant aberrance in voiding behavior (conscious cortical control) characterized by increased micturition frequency and reduced volume per micturition.Interestingly, remyelination restored healthy bladder function.However, there were no significant changes in the cystometric parameters (brainstem reflex) between the treatment groups.While MS is not classically considered a disease of aging, extending the longevity of these patients has not been reciprocated with improved treatments for their most-bothersome conditions, notably urinary symptoms that persist throughout life.Our data represent a novel compelling connection and strong correlation between CNS-myelination and cortical control of bladder function, which has potential implications in MS, aging, and aging-associated neurological disorders.

SENOLYTICS IN A MODEL OF ALZHEIMER'S DISEASE Ellen Wang, and Suckwon Lee, Buck Institute for Research on Aging, Novato, California, United States
The therapeutic effects of senescent cell killing with senolytics in neurodegeneration mouse models poise this strategy as an intervention candidate for Alzheimer's Disease (AD).However, it is unclear whether senolytic therapies for AD are translatable to human cells.To determine whether senolytics could be a viable therapeutic for AD, we have treated long-term mixed human neuron/astrocyte primary cultures with amyloid beta oligomers (ABO), which we have shown to induce a phenotype consistent with senescence in neurons.Fifteen days after ABO treatment, we administered Navitoclax (Nav) and the natural killer cell-line NK92, which are known to selectively kill senescent cells in the periphery.Following treatment, we assessed senescence markers in our cultures as well as senescent cell killing selectivity through cleaved Caspase 3 quantification.Our preliminary data show that Nav (8, 4, and 0.5uM) kills both control and ABO treated cells.NK92 cells (10 to 1 effector to target ratio) also kill some control cells, suggesting there is not a clear cut mechanism by which NK92 cells can distinguish senescent from non-senescent neurons or astrocytes.Although analysis of selective killing is ongoing, off-target killing indicates that we need more refined senolytic strategies to implement their safe human use.

SHORT-TERM DIESEL EXHAUST EXPOSURE RESULTS IN NEUROINFLAMMATION AND WHITE MATTER INJURY
Krista Lamorie-Foote, 1 Kristina Shkirkova, 2 Qinghai Liu, 2 Constantinos Sioutas, 2 Todd Morgan, 2 Caleb Finch, 2 and William Mack, 3 1.University of Southern California -Keck School of, Los Angeles, California, United States, 2. University of Southern California, Los Angeles, California, United States, 3. Keck School of Medicine of the University of Southern California, Los Angeles, California, United States Ambient air pollution (AAP) exposure is associated with white matter injury and cognitive decline in older adults (Chen et al. 2020,Erickson et al. 2020).Neuroinflammation and oxidative stress may contribute to this white matter injury.Diesel exhaust particulate matter (DEP) is a neurotoxic component of AAP.This study characterizes the time course by which neuroinflammation/oxidative stress occurs and results in white matter injury following DE exposure in a murine model.DEP (Sigma) was re-aerosolized for exposure.Mice were exposed to 100 µg/m3 DEP or filtered air (FA) for 5 hours (n=8/group), 100 hours (n=6/group), or 200 hours (n=6/group).Immunohistochemical analysis of degraded myelin basic protein (dMBP), a marker of myelin damage, was performed.Neuroinflammation and oxidative stress were assessed by histological analysis of complement C5a, an anaphylatoxin, and 4-Hydroxynonenal (4-HNE), a marker of lipid peroxidation.dMBPintegrated density was increased in the corpus callosum of DEP mice at 5 (p<0.01), 100 (p<0.01), and 200 hours (p<0.001) compared to FA mice.C5a integrated density was increased in the corpus callosum of DEP mice at 5 (p<0.01), 100 (p<0.01), and 200 hours (p<0.01)compared to FA mice.4-HNE integrated density was increased in the corpus callosum of DEP mice at 5 (p<0.001), 100 (p=0.001), and 200 hours (p<0.001) compared to FA mice.Neuroinflammation and oxidative stress are upregulated with associated white matter injury in the corpus callosum after 5 hours of DEP exposure.Short-term DEP exposure activates inflammatory/oxidative stress pathways, which may contribute to the pathogenesis of white matter injury.The lack of efficient medication against Alzheimer's disease (AD) is the most important problem for this health disorder today.One possible reason for this --the implementing medical interventions "too late in the disease stage" -has been recently addressed in the initiative that defined the preclinical AD stage by measuring changes in preclinical AD biomarkers.According to this definition, beta amyloid (Aβ) is one of the key preclinical AD biomarkers.Experimental studies showed that Aβ results from proteolytic cleavage of APP by β-and γ-secretases.Production of β-secretase involves BACE1 gene, activated by cellular stress response.This suggest that AD might be initiated by cellular stressors and that multifactorial regulation of AD is likely to be driven by genes involved in cellular stress response.In this paper we investigate whether interplay between SNPs from the EIF2AK4 gene involved in sensing cellular stress signals and the APP gene dealing with Aβ production may be associated with AD in human data.For this, we evaluated association of the interactions of the pairs of SNPs from these genes with AD in the analysis of HRS data.We found that interactions between several SNPs have statistically significant associations with AD.The results of this analysis confirm that the interplay between gene served as a sensor of cellular stress and gene involved in production of preclinical AD biomarker in response to Erickson et al. 2020,PMID:32182984; Chen  et al. 2020,PMID:32669395.