Mutant and curli-producing E. coli enhance the disease phenotype in a hSOD1-G93A mouse model of ALS.

The gut microbiome is a potential non-genetic contributing factor for Amyotrophic Lateral Sclerosis. Differences in gut microbial communities have been detected between ALS subjects and healthy controls, including an increase in Escherichia coli in ALS subjects. E. coli and other gram-negative bacteria produce curli proteins, which are functional bacterial amyloids. We examined whether long-term curli overexposure in the gut can exacerbate the development and progression of ALS. We utilized the slow-developing hSOD1-G93A mouse model of ALS with their C57BL/6J WT littermate controls, including males and females, with a total of 91 animals. These mice were on a normal chow diet and fed curli-producing or curli-nonproducing (mutant) E. coli in applesauce (vehicle) 3 times/week, from 1 through 7 months of age. Male hSOD1 mice demonstrated gradual slowing in running speed month 4 onwards, while females exhibited no signs of locomotive impairment even at 7 months of age. Around the same time, male hSOD1 mice showed a gradual increase in frequency of peripheral CD19+ B cells. Among the male hSOD1 group, chronic gut exposure to curli-producing E. coli led to significant shifts in α- and ß-diversities. Curli-exposed males showed suppression of immune responses in circulation, but an increase in markers of inflammation, autophagy and protein turnover in skeletal muscle. Some of these markers were also changed in mutant E. coli-exposed mice, including astrogliosis in the brainstem and demyelination in the lumbar spinal cord. Overall, chronic overexposure to a commensal bacteria like E. coli led to distant organ pathology in our model, without the presence of a leaky gut at 6 months. Mechanisms underlying gut-distant organ communication are of tremendous interest to all disciplines.

Loss of forebrain BIN1 attenuates hippocampal pathology and neuroinflammation in a tauopathy model.

Bridging integrator 1 (BIN1) is the second most prevalent genetic risk factor identified by genome-wide association studies (GWAS) for late-onset Alzheimer's disease. BIN1 encodes an adaptor protein that regulates membrane dynamics in the context of endocytosis and neurotransmitter vesicle release. In vitro evidence suggests that BIN1 can directly bind to tau in the cytosol. In addition, BIN1's function limits extracellular tau seed uptake by endocytosis and subsequent propagation as well as influences tau release through exosomes. However, the in vivo roles of BIN1 in tau pathogenesis and tauopathy-mediated neurodegeneration remain uncharacterized. We generated conditional knockout mice with a selective loss of Bin1 expression in the forebrain excitatory neurons and oligodendrocytes in P301S human tau transgenic background (line PS19). PS19 mice develop age-dependent tau neuropathology and motor deficits and are commonly used to study Alzheimer's disease tau pathophysiology. The severity of motor deficits and neuropathology was compared between experimental and control mice that differ with respect to forebrain BIN1 expression. BIN1's involvement in tau pathology and neuroinflammation was quantified by biochemical methods and immunostaining. Transcriptome changes were profiled by RNA-sequencing analysis to gain molecular insights. The loss of forebrain BIN1 expression in PS19 mice exacerbated tau pathology in the somatosensory cortex, thalamus, spinal cord and sciatic nerve, accelerated disease progression and caused early death. Intriguingly, the loss of BIN1 also mitigated tau neuropathology in select regions, including the hippocampus, entorhinal/piriform cortex, and amygdala, thus attenuating hippocampal synapse loss, neuronal death, neuroinflammation and brain atrophy. At the molecular level, the loss of forebrain BIN1 elicited complex neuronal and non-neuronal transcriptomic changes, including altered neuroinflammatory gene expression, concomitant with an impaired microglial transition towards the disease-associated microglial phenotype. These results provide crucial new information on in vivo BIN1 function in the context of tau pathogenesis. We conclude that forebrain neuronal BIN1 expression promotes hippocampal tau pathogenesis and neuroinflammation. Our findings highlight an exciting region specificity in neuronal BIN1 regulation of tau pathogenesis and reveal cell-autonomous and non-cell-autonomous mechanisms involved in BIN1 modulation of tau neuropathology.

Progression of Nonmotor Symptoms in Parkinson's Disease by Sex and Motor Laterality.

Nonmotor symptoms (NMS) in Parkinson's disease (PD) can start up to a decade before motor manifestations and strongly correlate with the quality of life. Understanding patterns of NMS can provide clues to the incipient site of PD pathology. Our goal was to systematically characterize the progression of NMS in PD (n = 489), compared to healthy controls, HC (n = 241), based on the sex of the subjects and laterality of motor symptom onset. Additionally, NMS experienced at the onset of PD were also compared to subjects with scans without dopaminergic deficit, SWEDD (n = 81). The Parkinson's Progression Markers Initiative (PPMI) database was utilized to analyze several NMS scales. NMS experienced by PD and SWEDD cohorts were significantly higher than HC and both sex and laterality influenced several NMS scales at the onset of motor symptoms. Sex Differences. PD males experienced significant worsening of sexual, urinary, sleep, and cognitive functions compared to PD females. PD females reported significantly increased thermoregulatory dysfunction and anxious mood over 7 years and significantly more constipation during the first 4 years after PD onset. Laterality Differences. At onset, PD subjects with right-sided motor predominance reported significantly higher autonomic dysfunction. Subjects with left-sided motor predominance experienced significantly more anxious mood at onset which continued as Parkinson's progressed. In conclusion, males experienced increased NMS burden in Parkinson's disease. Laterality of motor symptoms did not significantly influence NMS progression, except anxious mood. We analyzed NMS in a large cohort of PD patients, and these data are valuable to improve PD patients' quality of life by therapeutically alleviating nonmotor symptoms.

What Are the Molecular Mechanisms by Which Functional Bacterial Amyloids Influence Amyloid Beta Deposition and Neuroinflammation in Neurodegenerative Disorders?

Despite the enormous literature documenting the importance of amyloid beta (Ab) protein in Alzheimer's disease, we do not know how Ab aggregation is initiated and why it has its unique distribution in the brain. In vivo and in vitro evidence has been developed to suggest that functional microbial amyloid proteins produced in the gut may cross-seed Ab aggregation and prime the innate immune system to have an enhanced and pathogenic response to neuronal amyloids. In this commentary, we summarize the molecular mechanisms by which the microbiota may initiate and sustain the pathogenic processes of neurodegeneration in aging.

Diazepam Toxicity Presenting as a Dementia Disorder.

The toxicity associated with long-standing benzodiazepine use in older persons is a critical issue. Several epidemiological reports have studied correlation between benzodiazepine use and risk of dementia development. In this manuscript, we used a case report to demonstrate how chronic diazepam use can cause cognitive deficits that resemble Alzheimer's disease and related conditions. Benzodiazepine use is common in the geriatric population and is often taken for long periods of time in improper doses. In combination with age-related cortical atrophy on the MRI, our patient risked being misdiagnosed with Alzheimer's disease or another dementing disorder if not for the systematic investigation to resolve his symptoms. With elimination of the offending dispensable drug (diazepam), the patient's cognition improved greatly.

TAK1 regulates skeletal muscle mass and mitochondrial function.

Skeletal muscle mass is regulated by a complex array of signaling pathways. TGF-ß-activated kinase 1 (TAK1) is an important signaling protein, which regulates context-dependent activation of multiple intracellular pathways. However, the role of TAK1 in the regulation of skeletal muscle mass remains unknown. Here, we report that inducible inactivation of TAK1 causes severe muscle wasting, leading to kyphosis, in both young and adult mice.. Inactivation of TAK1 inhibits protein synthesis and induces proteolysis, potentially through upregulating the activity of the ubiquitin-proteasome system and autophagy. Phosphorylation and enzymatic activity of AMPK are increased, whereas levels of phosphorylated mTOR and p38 MAPK are diminished upon inducible inactivation of TAK1 in skeletal muscle. In addition, targeted inactivation of TAK1 leads to the accumulation of dysfunctional mitochondria and oxidative stress in skeletal muscle of adult mice. Inhibition of TAK1 does not attenuate denervation-induced muscle wasting in adult mice. Finally, TAK1 activity is highly upregulated during overload-induced skeletal muscle growth, and inactivation of TAK1 prevents myofiber hypertrophy in response to functional overload. Overall, our study demonstrates that TAK1 is a key regulator of skeletal muscle mass and oxidative metabolism.

Transmission Electron Microscopy for Analysis of Mitochondria in Mouse Skeletal Muscle.

Skeletal muscle is the most abundant tissue in the human body and regulates a variety of functions including locomotion and whole-body metabolism. Skeletal muscle has a plethora of mitochondria, the organelles that are essential for aerobic generation of ATP which provides the chemical energy to fuel vital functions such as contraction. The number of mitochondria in skeletal muscle and their function decline with normal aging and in various neuromuscular diseases and in catabolic conditions such as cancer, starvation, denervation, and immobilization. Moreover, compromised mitochondrial function is also associated with metabolic disorders including type 2 diabetes mellitus. It is now clear that maintaining mitochondrial content and function in skeletal muscle is vital for sustained health throughout the lifespan. While a number of staining methods are available to study mitochondria, transmission electron microscopy (TEM) is still the most important method to study mitochondrial structure and health in skeletal muscle. It provides critical information about mitochondrial content, cristae density, organization, formation of autophagosomes, and any other abnormalities commonly observed in various disease conditions. In this article, we describe a detailed protocol for sample preparation and analysis of mouse skeletal muscle mitochondria by TEM.

Emerging roles of ER stress and unfolded protein response pathways in skeletal muscle health and disease.

Skeletal muscle is the most abundant tissue in the human body and can adapt its mass as a consequence of physical activity, metabolism, growth factors, and disease conditions. Skeletal muscle contains an extensive network of endoplasmic reticulum (ER), called sarcoplasmic reticulum, which plays an important role in the regulation of proteostasis and calcium homeostasis. In many cell types, environmental and genetic factors that disrupt ER function cause an accumulation of misfolded and unfolded proteins in the ER lumen that ultimately leads to ER stress. To alleviate the stress and restore homeostasis, the ER activates a signaling network called the unfolded protein response (UPR). The UPR has three arms, which regulate protein synthesis and expression of many ER chaperone and regulatory proteins. However, the role of individual UPR pathways in skeletal muscle has just begun to be investigated. Recent studies suggest that UPR pathways play pivotal roles in muscle stem cell homeostasis, myogenic differentiation, and regeneration of injured skeletal muscle. Moreover, markers of ER stress and the UPR are activated in skeletal muscle in diverse conditions such as exercise, denervation, starvation, high fat diet, cancer cachexia, and aging. Accumulating evidence also suggests that ER stress may have important roles in the pathogenesis of inflammatory myopathies and genetic muscle disorders. The purpose of this review article is to discuss the role and potential mechanisms by which ER stress and the individual arms of the UPR regulate skeletal muscle formation, plasticity, and function in various physiological and pathophysiological conditions.

Isolation, Culturing, and Differentiation of Primary Myoblasts from Skeletal Muscle of Adult Mice.

Myogenesis is a multi-step process that leads to the formation of skeletal muscle during embryonic development and repair of injured myofibers. In this process, myoblasts are the main effector cell type which fuse with each other or to injured myofibers leading to the formation of new myofibers or regeneration of skeletal muscle in adults. Many steps of myogenesis can be recapitulated through in vitro differentiation of myoblasts into myotubes. Most laboratories use immortalized myogenic cells lines that also differentiate into myotubes. Although these cell lines have been found quite useful to delineating the regulatory mechanisms of myogenesis, they often show a great degree of variability depending on the origin of the cells and culture conditions. Primary myoblasts have been suggested as the most physiologically relevant model for studying myogenesis in vitro. However, due to their low abundance in adult skeletal muscle, isolation of primary myoblasts is technically challenging. In this article, we describe an improved protocol for the isolation of primary myoblasts from adult skeletal muscle of mice. We also describe methods for their culturing and differentiation into myotubes.

Distinct roles of TRAF6 and TAK1 in the regulation of adipocyte survival, thermogenesis program, and high-fat diet-induced obesity.

Chronic low-grade inflammation, adipocyte hypertrophy, and glucose intolerance are common features of obesity and a risk factor for cancer. Tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) is an adaptor protein that also possesses a non-conventional E3 ubiquitin ligase activity. In response to receptor-mediated events, TRAF6 activates transforming growth factor-activated kinase 1 (TAK1), which leads to activation of the MAPK and nuclear factor-kappa B (NF-κB) signaling pathways. However, the roles of TRAF6 and TAK1 in the regulation of adipocyte function remain less understood. Here, we demonstrate that adipocyte-specific deletion of TAK1, but not TRAF6, in mice reduces the survival of adipocytes and abundance of white adipose tissue (WAT). Adipocyte-specific ablation of TAK1, but not TRAF6, increases the expression for markers of beige/brown fat in WAT. Deletion of TAK1 in WAT increases phosphorylation of AMPK, abundance of PGC-1α, non-canonical NF-κB signaling, markers of M2 macrophages, and diminishes phosphorylation of JNK and canonical NF-κB signaling. Levels of TRAF6 and enzymatic activity of TAK1 are increased in WAT of mice fed with high-fat diet (HFD). Our results demonstrate that ablation of TAK1 drastically reduces HFD-induced obesity and improves energy expenditure and glucose metabolism. In contrast, adipocyte-specific ablation of TRAF6 has a minimal effect on HFD-induced obesity. Collectively, our results suggest that even though TRAF6 is an upstream activator of TAK1 in many signaling cascades, inactivation of TAK1, but not TRAF6, regulates adipocyte survival, energy expenditure, and HFD-induced obesity in mice.