Intercellular transmission of pathogenic proteins in ALS: Exploring the pathogenic wave.

In patients with amyotrophic lateral sclerosis (ALS), disease symptoms and pathology typically spread in a predictable spatiotemporal pattern beginning at a focal site of onset and progressing along defined neuroanatomical tracts. Like other neurodegenerative diseases, ALS is characterized by the presence of protein aggregates in postmortem patient tissue. Cytoplasmic, ubiquitin-positive aggregates of TDP-43 are observed in approximately 97% of sporadic and familial ALS patients, while SOD1 inclusions are likely specific to cases of SOD1-ALS. Additionally, the most common subtype of familial ALS, caused by a hexanucleotide repeat expansion in the first intron of the C9orf72 gene (C9-ALS), is further characterized by the presence of aggregated dipeptide repeat proteins (DPRs). As we will describe, cell-to-cell propagation of these pathological proteins tightly correlates with the contiguous spread of disease. While TDP-43 and SOD1 are capable of seeding protein misfolding and aggregation in a prion-like manner, C9orf72 DPRs appear to induce (and transmit) a 'disease state' more generally. Multiple mechanisms of intercellular transport have been described for all of these proteins, including anterograde and retrograde axonal transport, extracellular vesicle secretion, and macropinocytosis. In addition to neuron-to-neuron transmission, transmission of pathological proteins occurs between neurons and glia. Given that the spread of ALS disease pathology corresponds with the spread of symptoms in patients, the various mechanisms by which ALS-associated protein aggregates propagate through the central nervous system should be closely examined.

Chronic BMAA exposure combined with TDP-43 mutation elicits motor neuron dysfunction phenotypes in mice.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with an average age-of-onset of ∼60 years and is usually fatal within 2-5 years of diagnosis. Mouse models based upon single gene mutations do not recapitulate all ALS pathological features. Environmental insults may also contribute to ALS, and ß-N-methylamino-L-alanine (BMAA) is an environmental toxin linked with an increased risk of developing ALS. BMAA, along with cycasin, are hypothesized to be the cause of the Guam-ALS epicenter of the 1950s. We developed a multihit model based on low expression of a dominant familial ALS TDP-43 mutation (Q331K) and chronic low-dose BMAA exposure. Our two-hit mouse model displayed a motor phenotype absent from either lesion alone. By LC/MS analysis, free BMAA was confirmed at trace levels in brain, and were as high as 405 ng/mL (free) and 208 ng/mL (protein-bound) in liver. Elevated BMAA levels in liver were associated with dysregulation of the unfolded protein response (UPR) pathway. Our data represent initial steps towards an ALS mouse model resulting from combined genetic and environmental insult.

The influence of the thickness of non-piezoelectric pieces on pre-stressed piezotransducers.

The mechanical pre-stress applied in piezotransducers used to generate high power ultrasound is needed to avoid ceramics fracture on traction cycle. Pre-stress levels inferior to 50 MPa can yield resonance shifting due to effectiveness of acoustic coupling between transducer pieces. Symmetrical transducers with different thickness of passive parts were submitted to axial mechanical pre-stress up to 50 MPa and their resonances were measured. The experimental results show the increasing of the resonances frequencies with the level of applied pre-stress. Similar effect is verified in simulations by using a model based on Mason's equivalent electric circuit. Due to the similarity of these effects, a relation between applied pre-stress and pieces coupling was proposed for the transducer assembled. In addition, the dependence of the thickness of non-piezoelectric pieces on the coupling effectiveness between them is discussed. The results show that transducers with small thickness present more expressive shifting resonance ratio.

Nuclear calcium signaling controls CREB-mediated gene expression triggered by synaptic activity.

Information storage in the nervous system requires transcription triggered by synaptically evoked calcium signals. It has been suggested that translocation of calmodulin into the nucleus, initiated by submembranous calcium transients, relays synaptic signals to CREB. Here we show that in hippocampal neurons, signaling to CREB can be activated by nuclear calcium alone and does not require import of cytoplasmic proteins into the nucleus. The nucleus is particularly suited to integrate neuronal firing patterns, and specifies the transcriptional outputs through a burst frequency-to-nuclear calcium amplitude conversion. Calcium release from intracellular stores promotes calcium wave propagation into the nucleus, which is critical for CREB-mediated transcription by synaptic NMDA receptors. Pharmacological or genetic modulation of nuclear calcium may directly affect transcription-dependent memory and cognitive functions.

The resonance frequencies on mechanically pre-stressed ultrasonic piezotransducers.

The piezotransducers employed in high power ultrasound are composed of piezoelectric ceramics and metallic pieces. These transducers are mechanically pre-stressed in order to avoid the ceramic fractures when high voltage is applied under resonance. The resonance and anti-resonance frequencies are shifted depending on the level of applied mechanical pre-stressing. This paper discusses some causes of this shifting on a experimental study. The discussion takes into account the variations on characteristic parameters of the ceramics and the acoustic coupling between parts of the transducer.

The mechanical pre-stressing in ultrasonic piezotransducers.

Composed piezotransducers submitted to mechanical pre-stressing present shifts on resonance and anti-resonance frequencies. Changes on characteristic parameters of the ceramic and in the coupling between the parts of the transducer can be the causes for this behavior. In applications where the level of pre-stressing is low (up to 50 MPa) the parameters of the ceramic are not altered, therefore, the shifting on frequencies are attributed to coupling between parts. This paper describes a mathematical model to explain this effect based on difference of effective cross-section between transducers parts under pre-stressing. The results show a proportional relation between pre-stressing and effective coupling of the parts.

Results and cost analysis of distal [crural/pedal] arterial revascularisation for limb salvage in diabetic and non-diabetic patients.

In order to compare the outcome and costs of femorodistal grafting in diabetic and nondiabetic patients presenting with critical limb ischaemia we analysed a consecutive series of 109 femorodistal bypasses, 38 (35%) performed on people with diabetes and 71 (65%) on non-diabetic patients. The same aggressive revascularization policy was used in both groups with the decision to operate based on the presence of a calf or foot vessel on preoperative intra-arterial digital subtraction angiography (IADSA). Data were collected prospectively and the median follow-up was 15.4 months (range 0 to 42 months). There were no significant differences in 30-day (5.3% vs 4.2%) and in-hospital mortality (13.2% vs 14.1%) between the two groups. Life table curves at 3 years in diabetic and non-diabetic patients showed 48% vs 60% survival, 76% vs 72% knee salvage, 45% vs 56% limb salvage, and 38% vs 47% secondary patency. Although there was a trend for diabetic patients to perform less well, there was no statistically significant difference in these outcome measures. In cost comparison the only significant difference was found in the total hospital cost, which was Pounds 9181 in diabetic, compared to Pounds 6350 in nondiabetic patients (p = 0.026, Mann-Whitney). However, this cost was significantly less than that of primary amputation in either group (Pounds 15500 and Pounds 12040, respectively). Femorodistal reconstruction in both diabetic and non-diabetic patients, whenever feasible, is a cheaper option than primary amputation, even though vascular surgery may be more expensive in people with diabetes.

Consideraçöes sobre a produçäo de campos ultra-sônicos / Considerations about an ultrasonic field productions

O ultra-som é uma das principais ferramentas para diagnósticos na área médica. Através da interação dos campos ultra-sônicos pode-se obter várias características dos tecidos biológicos, portanto é importante conhecer sua distribuição de pressão no meio de propagação. Neste trabalho é estudado o campo ultra-sônico produzido por um transdutor piezoelétrico anular. Os resultados experimentais mostram que o campo ultra-sônico depende do material empregado na construção do transdutor.

The ultrasound is a important tool for diagnostics on medical area. Throught of ultrasonic fiel d interaction we can obtain some characteristics of the biologycal tissues, thus it is important to know the pressure distribuition in the propagation medium. ln this paper is studied the ultrasonic field generated by a ring piezoelectric transducer. The experimental results show the ultrasonic field depends of the material employed in the transducer construction.

Purification, pharmacological characterization and photoaffinity labeling of sigma receptors from rat and bovine brain.

The sigma receptor/binding site, found in the brain and periphery, binds haloperidol, (+)-benzomorphans, N-propyl-3-(3-hydroxyphenyl)-piperidine (3-PPP) and certain atypical neuroleptics with high affinity. We have succeeded in ca. 6,000-fold purification of protein(s) from rat and bovine cerebellum which display pharmacology characteristic of the sigma receptor. This purification was achieved by affinity chromatography using a Sepharose gel linked to a new high-affinity ligand, (S)-3-(3-methoxyphenyl)-3'-oxo-3'-phenyl-N-propylpiperidine, an analog of (S)-3-PPP. Elution of the affinity column with haloperidol afforded material which, after reconstitution into bimolecular lipid vesicles, was pharmacologically characterized by specific radioligand binding assays using [3H]haloperidol combined with competitive displacement using appropriate selective ligands. Comparison of the relative rank orders of potency of the ligands in these selective sigma receptor assays corresponded well with values obtained with tissue homogenates. The observed enantioselectivity for the binding of SKF-10,047 and cyclazocine suggests that the material purified corresponds to the sigma 1 receptor subtype. SDS-PAGE indicated that the purified material consisted of two bands of approximate molecular masses 65 and 63 kilodaltons. Photoaffinity labeling of the affinity-purified receptor with [3H]azido-DTG led to incorporation of the label into material of molecular mass 50-70 kDa, by slicing of SDS gels, while similar photolabeling of crude cerebellar homogenates led to exclusive labeling of a 29 kDa polypeptide, as found previously using other tissues. Molecular sizing under non-denaturing conditions indicated the photolabeled species is a labile large receptor complex of mass ca. 300-500 kDa which gradually breaks down upon standing at -80 degrees C into the lower mass (50-70 kDa) material. The sigma receptor ligand binding subunit, which appears to be of the sigma 1 subtype, appears to be contained within the 29 kDa polypeptide, which may be a subunit of the 63-65 kDa protein, which in turn appears to be a component of a much larger receptor complex. It further appears that the 29 kDa polypeptide is readily dissociable from a larger photolabeled sigma receptor complex in tissue homogenates, but does not dissociate from the photolabeled affinity-purified CHAPS-solubilized sigma receptor.

Expression of c-fos in regions of the basal limbic forebrain following intracerebroventricular corticotropin-releasing factor in unstressed or stressed male rats.

Corticotropin-releasing factor has an integrative role on the behavioral, endocrine and autonomic responses to stress. Immediate-early gene (c-fos) expression was used to determine patterns of neural activity in the limbic system following i.c.v. infusion of corticotropin-releasing factor. Either 250 or 1000 pmol corticotropin-releasing factor infused into the lateral ventricle of precannulated and handled male rats resulted in marked c-fos expression 60 or 120 min later in localized regions of the basal forebrain, including the ventrolateral septum, the dorsal and medial parvicellular divisions of the paraventricular nucleus, the central nucleus of the amygdala, and dorsal bed nucleus of the stria terminalis. Pre-infusion of alpha-helical corticotropin-releasing factor (2500 pmol), a competitive corticotropin-releasing factor antagonist of corticotropin-releasing factor, had no effect on immediate-early gene expression alone but reduced that elicited by exogenous i.c.v. corticotropin-releasing factor (250 pmol)--in some areas to control levels. Fifteen minutes of restraint stress, a situation in which corticotropin-releasing factor is released endogenously, also activated c-fos expression in a pattern that resembled corticotropin-releasing factor infusions but was not identical. There was enhanced expression in the dorsal and medial areas of the paraventricular nucleus, but not its magnocellular region, and increased expression in the ventrolateral septum; however, there was no detectable response on the central amygdala. Preinfusion of alpha-helical corticotropin-releasing factor (2500 pmol) had no significant effect on stress-induced c-fos expression in the ventrolateral septum or paraventricular nucleus. This suggests that corticotropin-releasing factor release may form only a part of the central neurochemical response to restraint stress. Rats given i.c.v. corticotropin-releasing factor (250 pmol) before restraint stress showed additive effects on c-fos in the ventrolateral septum but not in the paraventricular nucleus; the central nucleus of the amygdala reacted as if corticotropin-releasing factor alone had been infused. Corticosterone levels were raised by both stress and corticotropin-releasing factor, but pretreatment with alpha-helical corticotropin-releasing factor reduced them after either procedure, which correlates with c-fos expression in the paraventricular nucleus and ventrolateral septum. These results show that corticotropin-releasing factor induces a specific pattern of c-fos expression in localized regions of the amygdala, hypothalamus and septum, which may indicate a corresponding pattern of neural activation. Restraint, one form of stress, activates c-fos in a similar but not identical manner, suggesting that corticotropin-releasing factor may not be the only neuropeptide involved in the response to this stressor.(ABSTRACT TRUNCATED AT 400 WORDS)

Decreased sensitivity to metocurine during long-term phenytoin therapy may be attributable to protein binding and acetylcholine receptor changes.

Long-term phenytoin therapy induces resistance to the neuromuscular blocking effects of metocurine. The hypothesis that this is attributable to increased plasma protein binding of the drug (decreased free fraction) related to increased concentrations of alpha 1-acid glycoprotein (AAG) or attributable to the proliferation of acetylcholine receptors (AChR) at the muscle membrane was tested in the rat. After 14 days of phenytoin 40 mg.kg-1, administered intraperitoneally twice daily (n = 12), the neuromuscular pharmacodynamics were evaluated and compared with those of time-matched controls (n = 10). Protein binding was measured by equilibrium dialysis, AAG concentrations by radial immunodiffusion assay, and AChR by 125I-alpha-bungarotoxin binding. The effective dose for 50% inhibition of baseline twitch height (ED50) was significantly greater in the phenytoin group than in the control group (15.03 +/- 1.65 micrograms.kg-1 vs. 9.98 +/- 0.69 micrograms.kg-1, respectively). The concentrations of AAG increased gradually from 133.8 +/- 7.8 micrograms.ml-1 at day 0, to 343.1 +/- 58.0 micrograms.ml-1 at day 7, to 1,729.5 +/- 422.3 micrograms.ml-1 at day 14 in the phenytoin group. The induction of AAG concentrations in plasma was dependent on plasma phenytoin concentrations and was most prominent after 14 days of phenytoin (r = 0.77; P less than 0.01; n = 22). The free fraction of metocurine was significantly decreased in the phenytoin group compared to the control group (67.2 +/- 0.18% vs. 74.5 +/- 2.5%). There was a significant negative correlation between increased AAG concentrations and decreased free fraction (r = 0.65).(ABSTRACT TRUNCATED AT 250 WORDS)

Radial immunodiffusion assay for rat alpha 1-acid glycoprotein.

alpha 1-Acid glycoprotein (AGP) is an "acute phase protein" whose expression is altered in several human pathologies. Using antiserum from New Zealand white rabbits, a radial immunodiffusion assay for measuring AGP levels in rat plasma was developed operating in the range of 50-2500 micrograms/ml with high specificity. Standard curves were constructed (precipitin ring diameter 2 vs. micrograms/ml AGP) yielding highly linear plots (r = .98). The plasma concentration of AGP in spontaneously hypertensive (SHR) rats was double that of the normotensive Kyoto-Wistar (WKY) rats (208 +/- 10 vs. 118 +/- 5 micrograms/ml). AGP induction by turpentine resulted in a 14- and 26-fold increase in AGP levels in SHR and WKY rats, respectively. Induction of AGP by dexamethasone injection was examined in the SHR and WKY rat strains resulting in a 5- and 12-fold increase in AGP levels, respectively. AGP concentration in whole brain of rats was determined to be 12.7 +/- 1.8 micrograms/g. AGP concentrations in SHR and WKY liver were also determined to be 159 +/- 3 and 148 +/- 5 micrograms/g liver tissue.

Olfactory bulbectomy alters alpha-1 acid glycoprotein levels in rat plasma.

Olfactory bulbectomy in rats causes neurochemical, behavioral, as well as physiological alterations. These alterations make this surgical procedure a useful animal model for depression. In humans, depression was shown to be accompanied by increases in plasma cortisol, inability to decrease cortisol in the dexamethasone suppression test and increases in plasma alpha-1 acid glycoprotein (AGP), an endogenous modulator for the serotonin uptake site. Utilizing a recently developed radial immunodiffusion assay for rat AGP we were able to confirm the increases in plasma AGP in the rat. However, we did not observe increased corticosterone in the rat. We also observed the aggressive behavior of muricide in olfactory bulbectomized rats. These results seem to indicate that olfactory bulbectomy is a good model for depression in the human condition and that AGP may be a putative marker for this condition.