Improvement of Impaired Memory in Mice by Taurine

Taurine was extracted from Pegasus later-narius Cuvier to study its effects on learning and memory in mice. Mice were treated with different doses of taurine (10 mg/kg, 20 mg/kg, 40 mg/kg). The mice were treated with various chemical agents (pentobarbital, cycloheximide, sodium nitrite, alcohol) to disrupt the normal memory process. We measured the effect of taurine on step-down latency (SDL) and escape latency (EL) in a passive avoidance task after 10 or 30 days. Treatment with taurine alone did not change either SDL or EL. Taurine protected mice .from the memory disruption induced by alcohol, pentobarbital, sodium nitrite, and cycloheximide but had no obvious effect on motor coordination, exploratory activity, or locomotor activity as measured using the rota-rod test and the hole board test. We conclude that taurine can be effective in attenuating the amnesia produced by alcohol, pentobarbital, cycloheximide, and sodium nitrite without compromising the behavioral aspects of the animals tested

Differential Regional and Subtype-Specific Vulnerability of Enteric Neurons to Mitochondrial Dysfunction

Mitochondrial dysfunction is a central mediator of disease progression in diverse neurodegenerative diseases that often present with prominent gastrointestinal abnormalities. Gastrointestinal dysfunction in these disorders is related, at least in part, to defects in the enteric nervous system (ENS). The role of mitochondrial deficits in ENS neurodegeneration and their relative contribution to gastrointestinal dysfunction, however, are unclear. To better understand how mitochondrial abnormalities in the ENS influence enteric neurodegeneration and affect intestinal function, we generated mice (Tfam-ENSKOs) with impaired mitochondrial metabolism in enteric neurons and glia through the targeted deletion of the mitochondrial transcription factor A gene (Tfam). Tfam-ENSKO mice were initially viable but, at an early age, they developed severe gastrointestinal motility problems characterized by intestinal pseudo-obstruction resulting in premature death. This gastrointestinal dysfunction was caused by extensive, progressive neurodegeneration of the ENS involving both neurons and glia. Interestingly, mitochondrial defects differentially affected specific subpopulations of enteric neurons and regions of the gastrointestinal tract. Mitochondrial deficiency-related neuronal and glial loss was most prominent in the proximal small intestine, but the first affected neurons, nitrergic inhibitory neurons, had the greatest losses in the distal small intestine. This regional and subtype-specific variability in susceptibility to mitochondrial defects resulted in an imbalance of inhibitory and excitatory neurons that likely accounts for the observed phenotype in Tfam-ENSKO mice. Mitochondrial dysfunction, therefore, is likely to be an important driving force of neurodegeneration in the ENS and contribute to gastrointestinal symptoms in people with neurodegenerative disorders

CNP-Cre excises Tfam in enteric neurons and glia and disrupts ENS mitochondria in Tfam-ENSKO mice.

<p><b><i>a</i></b><i>)</i> YFP fluorescence in 2-day-old Rosa26-YFP/CNP-Cre mice overlaps (merge) with enteric neurons (HuC/HuD⁺ cells, Hu, red) and glia (Sox-10⁺ cells, Sox, blue). Excision-dependent YFP fluorescence is visible in the majority of enteric neurons and glia in all gut regions. Asterisks indicate the occasional non-recombined, non-YFP⁺ cell. Scale bars: 30 µm. <b><i>b</i></b><i>)</i> Quantification of the percent of YFP⁺ neurons (% of HuC/HuD⁺ and YFP⁺ cells/total HuC/HuD⁺ cells) and glia (% of Sox-10⁺ and YFP⁺ cells/total Sox-10⁺ cells) in different gut regions confirms high efficiency of CNP-Cre mediated recombination in the ENS. n = 3 mice per genotype. <b><i>c</i></b><i>)</i> RT-PCR of Tfam transcript demonstrates the complete excision of <i>Tfam</i> in YFP⁺ FACS selected myenteric neurons and glia in YFP/Tfam-ENSKO mice at 7 weeks of age. n = 3 mice per genotype. <b><i>d</i></b><i>)</i> qRT-PCR results show depletion of mtDNA content in YFP⁺ FACS-selected myenteric neurons and glia in 7 week old YFP/Tfam-ENSKO mice. mtDNA content was significantly reduced in YFP/Tfam-ENSKO mice in all regions examined at this age (*, p<0.01). Reported values are normalized to nuclear DNA content and ratio of mtDNA to nuclear DNA in control animals was set at 100; n = 3 mice per genotype. <b><i>e</i></b><i>)</i> Electron micrographs of myenteric plexus mitochondria (arrowheads) in 7 week old control and Tfam-ENSKO mice. Tfam-ENSKO myenteric neurons and glia contain abundant abnormal, enlarged mitochondria with dilated and distorted cristae. Scale bars, 500 nm.</p

Villus Neurite Density.

<p>Density of neuronal projections in intestinal villi in 2 and 7 week old Tfam-ENSKO and control mice. n = 3 for each genotype and age. PSI: proximal small intestine, DSI: distal small intestine, Co: colon.</p

Early and differential loss of nitrergic inhibitory neurons in Tfam-ENSKO mice.

<p><b><i>a</i></b><i>)</i> Representative images of NADPH-d stained myenteric plexus show inhibitory neurons in the proximal SI and distal SI in 7 week old Tfam-ENSKO mice. Scale bar, 150 µm. <b><i>b</i></b><i>)</i> Quantitative analysis of NADPH-d⁺ neuron density in Tfam-ENSKOs relative to control littermates at 2 and 7 weeks of age. n = 3 for each genotype at each age. <b><i>c</i></b><i>)</i> Ratio of nitrergic neurons to total neurons in 7 week old Tfam-ENSKO and control mice. n = 3 for each genotype. PSI: proximal small intestine, DSI: distal small intestine, Co: colon. <b><i>d</i></b><i>)</i> Diagram depicting how the imbalance of inhibitory neurons to excitatory neurons in each region in Tfam-ENSKOs could produce the observed proximal SI dilation and distal SI constriction. In Tfam-ENSKO mice, greater total neuron loss (Tfam-ENSKO top panel) relative to NADPH-d⁺ neuron loss (Tfam- ENSKO middle panel) would result in increased inhibitory input and dilation of the proximal SI (Tfam-ENSKO bottom panel). Greater NADPH-d⁺ neuron loss (Tfam-ENSKO middle panel) relative to total neuron loss (Tfam-ENSKO top panel) would result in decreased inhibitory input and constriction (and pseudoobstruction) of the distal SI of Tfam-ENSKO mice (bottom panel). Color intensity represents neuronal density with WT density set as the most intense color in all regions of the bowel.</p

<i>In vitro</i> Mitochondrial Inhibitor Assay Results.

<p><i>In vitro</i> mitochondrial inhibitor assay results from cultured E12.5 immunoselected enteric neurons. n =  duplicate wells for each condition from 3 independent assays. Veh = Vehicle; Rot = Rotenone; Antim = Antimycin</p

Tfam-ENSKO mice display progressive degeneration of neurons and glia with distinct regional vulnerabilities.

<p><b><i>a</i></b><i>)</i> Representative images of HuC/HuD (neurons, red) and Sox-10 (glia, green) immunohistochemistry in three regions of myenteric plexus of 7 week old control and Tfam-ENSKO mice. Loss of both HuC/HuD⁺ neurons and Sox-10⁺ glia is apparent in Tfam-ENSKOs at this age. Scale bars, 100 µm. <b><i>b</i></b><i>)</i> Quantification of total myenteric neuron density in Tfam-ENSKOs expressed relative to control littermates at 2 and 7 weeks of age. For 2 weeks old, n = 6 (Ctrl) and n = 4 (Tfam-ENSKO). For 7 weeks old, n = 3 for each genotype. <b><i>c</i></b><i>)</i> Quantification of glial density in Tfam-ENSKOs expressed relative to control littermates at 2 and 7 weeks of age. For 2 weeks old, n = 6 (Ctrl) and n = 4 (Tfam-ENSKO). For 7 weeks old, n = 3 for each genotype. <b><i>d</i></b><i>)</i> Neuron-to-glia ratio in ENS of Tfam-ENSKOs and control littermates at 2 and 7 weeks of age demonstrates that relative cell loss is equivalent for both neurons and glia. For 2 weeks old, n = 6 (Ctrl) and n = 4 (Tfam-ENSKO). For 7 weeks old, n = 3 for each genotype. PSI: proximal small intestine, DSI: distal small intestine, Co: colon.</p

Myenteric Plexus Cell Density.

<p>Myenteric plexus neuron and glial cell density and ratios in 2 and 7-week-old Tfam-ENSKO and control mice. n = 3 for each genotype for all analyses of 7 week old mice. For 2 week old mice, n = 6 (Ctrl) or 4 (Tfam-ENSKO) for total neuron density, glial density, and neuron to glia ratio and n = 3 for NADPH-d⁺ neuron density. N/A, not applicable; due to small size of tissue samples at 2 weeks of age, staining for both HuC/HuD⁺ (total) and NADPH-d⁺ (nitrergic) could not be performed on a single animal and therefore statistical analysis of the proportion of total neurons that are NADPH-d⁺ was not possible. PSI: proximal small intestine, DSI: distal small intestine, Co: colon.</p

Nitrergic Fiber Density.

<p>Myenteric NADPH-d⁺ fiber density and number of NADPH-d⁺ fibers per neuron in 7 week old Tfam-ENSKO and control mice. n = 3 for each genotype. PSI: proximal small intestine, DSI: distal small intestine, Co: colon.</p