MATR3 P154S knock-in mice do not exhibit motor, muscle or neuropathologic features of ALS.

Matrin 3 is a nuclear matrix protein that has many roles in RNA processing including splicing and transport of mRNA. Many missense mutations in the Matrin 3 gene (MATR3) have been linked to familial forms of amyotrophic lateral sclerosis (ALS) and distal myopathy. However, the exact role of MATR3 mutations in ALS and myopathy pathogenesis is not understood. To demonstrate a role of MATR3 mutations in vivo, we generated a novel CRISPR/Cas9 mediated knock-in mouse model harboring the MATR3 P154S mutation expressed under the control of the endogenous promoter. The P154S variant of the MATR3 gene has been linked to familial forms of ALS. Heterozygous and homozygous MATR3 P154S knock-in mice did not develop progressive motor deficits compared to wild-type mice. In addition, ALS-like pathology did not develop in nervous or muscle tissue in either heterozygous or homozygous mice. Our results suggest that the MATR3 P154S variant is not sufficient to produce ALS-like pathology in vivo.

Glucocorticoids exacerbate cognitive deficits in TDP-25 transgenic mice via a glutathione-mediated mechanism: implications for aging, stress and TDP-43 proteinopathies.

The accumulation of TDP-43 (transactive response DNA-binding protein 43) and its 25 kDa C-terminal fragment (TDP-25) is a hallmark of several neurodegenerative disorders, including frontotemporal lobar degeneration (FTLD-TDP) and amyotrophic lateral sclerosis (ALS). The majority of FTLD-TDP cases are due to loss of function mutations in the gene encoding progranulin, a secreted growth factor. In ALS, specific mutations in the gene encoding TDP-43 have been linked to the disease pathogenesis. In both cases, however, the penetrance of the mutations greatly increases during aging, suggesting that other genetic or environmental factors may facilitate the development of the disease. Using transgenic mice that overexpress the 25 kDa C-terminal fragment of TDP-43, here we show that glucocorticoids, stress hormones known to increase the brain susceptibility to neurotoxic insults, increase the levels of soluble TDP-25 and exacerbate cognitive deficits, without altering full-length TDP-43 levels. Additionally, we show that the mechanism underlying the glucocorticoid-mediated increase in TDP-25 levels is coupled to changes in the glutathione redox state. Glutathione is an antioxidant involved in protecting cells from damage caused by reactive oxygen species; notably, alterations in the ratio of reduced to oxidized glutathione, which is the primary determinant of the cellular redox state, are associated with aging and neurodegeneration. We show that restoring the ratio of reduced to oxidized glutathione blocks the glucocorticoid effects on TDP-25. These data show that glucocorticoids potentiate the neurotoxic action of TDP-25 by increasing its levels and clearly indicate the role of cellular oxidative damage in this process.

Naturally secreted amyloid-beta increases mammalian target of rapamycin (mTOR) activity via a PRAS40-mediated mechanism.

Reducing the mammalian target of rapamycin (mTOR) activity increases lifespan and health span in a variety of organisms. Alterations in protein homeostasis and mTOR activity and signaling have been reported in several neurodegenerative disorders, including Alzheimer disease (AD); however, the causes of such deregulations remain elusive. Here, we show that mTOR activity and signaling are increased in cell lines stably transfected with mutant amyloid precursor protein (APP) and in brains of 3xTg-AD mice, an animal model of AD. In addition, we show that in the 3xTg-AD mice, mTOR activity can be reduced to wild type levels by genetically preventing Aß accumulation. Similarly, intrahippocampal injections of an anti-Aß antibody reduced Aß levels and normalized mTOR activity, indicating that high Aß levels are necessary for mTOR hyperactivity in 3xTg-AD mice. We also show that the intrahippocampal injection of naturally secreted Aß is sufficient to increase mTOR signaling in the brains of wild type mice. The mechanism behind the Aß-induced mTOR hyperactivity is mediated by the proline-rich Akt substrate 40 (PRAS40) as we show that the activation of PRAS40 plays a key role in the Aß-induced mTOR hyperactivity. Taken together, our data show that Aß accumulation, which has been suggested to be the culprit of AD pathogenesis, causes mTOR hyperactivity by regulating PRAS40 phosphorylation. These data further indicate that the mTOR pathway is one of the pathways by which Aß exerts its toxicity and further support the idea that reducing mTOR signaling in AD may be a valid therapeutic approach.

Longitudinal evaluation of myofiber microstructural changes in a preclinical ALS model using the transverse relaxivity at tracer equilibrium (TRATE): A preliminary study.

T2⁎ relaxivity contrast imaging may serve as a potential imaging biomarker for amyotrophic lateral sclerosis (ALS) by noninvasively quantifying the tissue microstructure. In this preliminary longitudinal study, we investigated the Transverse Relaxivity at Tracer Equilibrium (TRATE) in three muscle groups between SOD1-G93A (ALS model) rat and a control population at two different timepoints. The control group was time matched to the ALS group such that the second timepoint was the onset of disease. We observed a statistically significant decrease in TRATE over time in the gastrocnemius, tibialis, and digital flexor muscles in the SOD1-G93A model (p-value = 0.003, 0.008, 0.005; respectively), whereas TRATE did not change over time in the control group (p-value = 0.4777, 0.6837, 0.9682; respectively). Immunofluorescent staining revealed a decrease in minimum fiber area and cell density in the SOD1-G93A model when compared to the control group (p-value = 6.043E-10 and 2.265E-10, respectively). These microstructural changes observed from histology align with the theorized biophysical properties of TRATE. We demonstrate that TRATE can longitudinally differentiate disease associated atrophy from healthy muscle and has potential to serve as a biomarker for disease progression and ultimately therapy response in patients with ALS.

Intravenously Administered, Retinoid Activating Nanoparticles Increase Lifespan and Reduce Neurodegeneration in the SOD1G93A Mouse Model of ALS.

Dysregulation of the retinoic acid (RA) signaling pathway is observed in amyotrophic lateral sclerosis (ALS) and other neurodegenerative disorders. Here, we investigated the therapeutic potential of retinoid activation via the RA receptor ß (RARß) in the SOD1 G93A mouse model of ALS. Our approach utilized the RARß agonist adapalene, which we previously found to be neuroprotective in vitro. Adapalene, like most retinoids, is poorly water soluble, which has thus far prevented effective drug delivery in vivo. To address this challenge, we encapsulated adapalene within nanoparticles (Adap-NPs) composed of poly(lactic acid)-poly(ethylene glycol) (PLA-PEG). Our data demonstrate that intravenous administration of Adap-NPs robustly activates retinoid signaling in the CNS. Chronic administration of Adap-NPs resulted in improved motor performance, prolonged lifespan, and neuroprotection in SOD1 G93A mice. This study highlights retinoid signaling as a valuable therapeutic approach and presents a novel nanoparticle platform for the treatment of ALS.

Generation of two induced pluripotent stem cell (iPSC) lines from an ALS patient with simultaneous mutations in KIF5A and MATR3 genes.

Fibroblasts from an amyotrophic lateral sclerosis patient with simultaneous mutations in the MATR3 gene and KIF5A gene were isolated and reprogrammed into induced pluripotent stem cells via a non-integrating Sendai viral vector. The generated iPSC clones demonstrated normal karyotype, expression of pluripotency markers, and the capacity to differentiate into three germ layers. The unique presence of two simultaneous mutations in ALS-associated genes represent a novel tool for the study of ALS disease mechanisms.

Optical barcoding of PLGA for multispectral analysis of nanoparticle fate in vivo.

Understanding of the mechanisms by which systemically administered nanoparticles achieve delivery across biological barriers remains incomplete, due in part to the challenge of tracking nanoparticle fate in the body. Here, we develop a new approach for "barcoding" nanoparticles composed of poly(lactic-co-glycolic acid) (PLGA) with bright, spectrally defined quantum dots (QDs) to enable direct, fluorescent detection of nanoparticle fate with subcellular resolution. We show that QD labeling does not affect major biophysical properties of nanoparticles or their interaction with cells and tissues. Live cell imaging enabled simultaneous visualization of the interaction of control and targeted nanoparticles with bEnd.3 cells in a flow chamber, providing direct evidence that surface modification of nanoparticles with the cell-penetrating peptide TAT increases their biophysical association with cell surfaces over very short time periods under convective current. We next developed this technique for quantitative biodistribution analysis in vivo. These studies demonstrate that nanoparticle surface modification with the cell penetrating peptide TAT facilitates brain-specific delivery that is restricted to brain vasculature. Although nanoparticle entry into the healthy brain parenchyma is minimal, with no evidence for movement of nanoparticles across the blood-brain barrier (BBB), we observed that nanoparticles are able to enter to the central nervous system (CNS) through regions of altered BBB permeability - for example, into circumventricular organs in the brain or leaky vasculature of late-stage intracranial tumors. In sum, these data demonstrate a new, multispectral approach for barcoding PLGA, which enables simultaneous, quantitative analysis of the fate of multiple nanoparticle formulations in vivo.

Accumulation of C-terminal fragments of transactive response DNA-binding protein 43 leads to synaptic loss and cognitive deficits in human TDP-43 transgenic mice.

Accumulation of the transactive response DNA-binding protein 43 (TDP-43) is a major hallmark of several neurodegenerative disorders, collectively known as TDP-43 proteinopathies. The most common TDP-43 proteinopathies, frontotemporal lobar degeneration with TDP-43-positive inclusions, and amyotrophic lateral sclerosis, share overlapping neuropathological and clinical phenotypes. The development and detailed analysis of animal models of TDP-43 proteinopathies are critical for understanding the pathogenesis of these disorders. Transgenic mice overexpressing mutant human TDP-43 (herein referred to as hTDP-43) are characterized by neurodegeneration and reduced life span. However, little is known about the behavioral phenotype of these mice. Here we report the novel finding that hTDP-43 mice develop deficits in cognition, motor performance, and coordination. We show that these behavioral deficits are associated with the accumulation of nuclear and cytosolic TDP-43 C-terminal fragments, a decrease in endogenous TDP-43 levels, and synaptic loss. Our findings provide critical insights into disease pathology, and will help guide future preclinical studies aimed at testing the effects of potential therapeutic agents on the onset and progression of TDP-43 proteinopathies.

Amyloid beta and the longest-lived rodent: the naked mole-rat as a model for natural protection from Alzheimer's disease.

Amyloid beta (Aß) is implicated in Alzheimer's disease (AD) as an integral component of both neural toxicity and plaque formation. Brains of the longest-lived rodents, naked mole-rats (NMRs) approximately 32 years of age, had levels of Aß similar to those of the 3xTg-AD mouse model of AD. Interestingly, there was no evidence of extracellular plaques, nor was there an age-related increase in Aß levels in the individuals examined (2-20+ years). The NMR Aß peptide showed greater homology to the human sequence than to the mouse sequence, differing by only 1 amino acid from the former. This subtle difference led to interspecies differences in aggregation propensity but not neurotoxicity; NMR Aß was less prone to aggregation than human Aß. Nevertheless, both NMR and human Aß were equally toxic to mouse hippocampal neurons, suggesting that Aß neurotoxicity and aggregation properties were not coupled. Understanding how NMRs acquire and tolerate high levels of Aß with no plaque formation could provide useful insights into AD, and may elucidate protective mechanisms that delay AD progression.

mTOR regulates tau phosphorylation and degradation: implications for Alzheimer's disease and other tauopathies.

Accumulation of tau is a critical event in several neurodegenerative disorders, collectively known as tauopathies, which include Alzheimer's disease and frontotemporal dementia. Pathological tau is hyperphosphorylated and aggregates to form neurofibrillary tangles. The molecular mechanisms leading to tau accumulation remain unclear and more needs to be done to elucidate them. Age is a major risk factor for all tauopathies, suggesting that molecular changes contributing to the aging process may facilitate tau accumulation and represent common mechanisms across different tauopathies. Here, we use multiple animal models and complementary genetic and pharmacological approaches to show that the mammalian target of rapamycin (mTOR) regulates tau phosphorylation and degradation. Specifically, we show that genetically increasing mTOR activity elevates endogenous mouse tau levels and phosphorylation. Complementary to it, we further demonstrate that pharmacologically reducing mTOR signaling with rapamycin ameliorates tau pathology and the associated behavioral deficits in a mouse model overexpressing mutant human tau. Mechanistically, we provide compelling evidence that the association between mTOR and tau is linked to GSK3ß and autophagy function. In summary, we show that increasing mTOR signaling facilitates tau pathology, while reducing mTOR signaling ameliorates tau pathology. Given the overwhelming evidence that reducing mTOR signaling increases lifespan and healthspan, the data presented here have profound clinical implications for aging and tauopathies and provide the molecular basis for how aging may contribute to tau pathology. Additionally, these results provide preclinical data indicating that reducing mTOR signaling may be a valid therapeutic approach for tauopathies.

Lifelong rapamycin administration ameliorates age-dependent cognitive deficits by reducing IL-1ß and enhancing NMDA signaling.

Understanding the factors that contribute to age-related cognitive decline is imperative, particularly as age is the major risk factor for several neurodegenerative disorders. Levels of several cytokines increase in the brain during aging, including IL-1ß, whose levels positively correlate with cognitive deficits. Previous reports show that reducing the activity of the mammalian target of rapamycin (mTOR) extends lifespan in yeast, nematodes, Drosophila, and mice. It remains to be established, however, whether extending lifespan with rapamycin is accompanied by an improvement in cognitive function. In this study, we show that 18-month-old mice treated with rapamycin starting at 2 months of age perform significantly better on a task measuring spatial learning and memory compared to age-matched mice on the control diet. In contrast, rapamycin does not improve cognition when given to 15-month-old mice with pre-existing, age-dependent learning and memory deficits. We further show that the rapamycin-mediated improvement in learning and memory is associated with a decrease in IL-1ß levels and an increase in NMDA signaling. This is the first evidence to show that a small molecule known to increase lifespan also ameliorates age-dependent learning and memory deficits.

Methylene blue reduces aß levels and rescues early cognitive deficit by increasing proteasome activity.

Promising results have emerged from a phase II clinical trial testing methylene blue (MB) as a potential therapeutic for Alzheimer disease (AD), where improvements in cognitive functions of AD patients after 6 months of MB administration have been reported. Despite these reports, no preclinical testing of MB in mammals has been published, and thus its mechanism of action in relation to AD pathology remains unknown. In order to elucidate the effects of MB on AD pathology and to determine its mechanism of action, we used a mouse model (3xTg-AD) that develops age-dependent accumulation of Aß and tau and cognitive decline. Here, we report that chronic dietary MB treatment reduces Aß levels and improves learning and memory deficits in the 3xTg-AD mice. The mechanisms underlying the effects of MB on Aß pathology appears to be mediated by an increase in Aß clearance as we show that MB increases the chymotrypsin- and trypsin-like activities of the proteasome in the brain. To our knowledge, this is the first report showing that MB increases proteasome function and ameliorates AD-like pathology in vivo. Overall, the data presented here support the use of MB for the treatment of AD and offer a possible mechanism of action.