Larisa Ryskalin

Compartment-dependent mitochondrial alterations in experimental ALS, the effects of mitophagy and mitochondriogenesis

Amyotrophic lateral sclerosis (ALS) is characterized by massive loss of motor neurons. Data from ALS patients and experimental models indicate that mitochondria are severely damaged within dying or spared motor neurons. Nonetheless, recent data indicate that mitochondrial preservation, although preventing motor neuron loss, fails to prolong lifespan. On the other hand, the damage to motor axons plays a pivotal role in determining both lethality and disease course. Thus, in the present article each motor neuron compartment (cell body, central, and peripheral axons) of G93A SOD-1 mice was studied concerning mitochondrial alterations as well as other intracellular structures. We could confirm the occurrence of ALS-related mitochondrial damage encompassing total swelling, matrix dilution and cristae derangement along with non-pathological variations of mitochondrial size and number. However, these alterations occur to a different extent depending on motor neuron compartment. Lithium, a well-known autophagy inducer, prevents most pathological changes. However, the efficacy of lithium varies depending on which motor neuron compartment is considered. Remarkably, some effects of lithium are also evident in wild type mice. Lithium is effective also in vitro, both in cell lines and primary cell cultures from the ventral spinal cord. In these latter cells autophagy inhibition within motor neurons in vitro reproduced ALS pathology which was reversed by lithium. Muscle and glial cells were analyzed as well. Cell pathology was mostly severe within peripheral axons and muscles of ALS mice. Remarkably, when analyzing motor axons of ALS mice a subtotal clogging of axoplasm was described for the first time, which was modified under the effects of lithium. The effects induced by lithium depend on several mechanisms such as direct mitochondrial protection, induction of mitophagy and mitochondriogenesis. In this study, mitochondriogenesis induced by lithium was confirmed in situ by a novel approach using [2-3H]-adenosine.

mTOR-Dependent Cell Proliferation in the Brain

The mammalian Target of Rapamycin (mTOR) is a molecular complex equipped with kinase activity which controls cell viability being key in the PI3K/PTEN/Akt pathway. mTOR acts by integrating a number of environmental stimuli to regulate cell growth, proliferation, autophagy, and protein synthesis. These effects are based on the modulation of different metabolic pathways. Upregulation of mTOR associates with various pathological conditions, such as obesity, neurodegeneration, and brain tumors. This is the case of high-grade gliomas with a high propensity to proliferation and tissue invasion. Glioblastoma Multiforme (GBM) is a WHO grade IV malignant, aggressive, and lethal glioma. To date, a few treatments are available although the outcome of GBM patients remains poor. Experimental and pathological findings suggest that mTOR upregulation plays a major role in determining an aggressive phenotype, thus determining relapse and chemoresistance. Among several activities, mTOR-induced autophagy suppression is key in GBM malignancy. In this article, we discuss recent evidence about mTOR signaling and its role in normal brain development and pathological conditions, with a special emphasis on its role in GBM.

A rare genetic variant of BPIFB4 predisposes to high blood pressure via impairment of nitric oxide signaling

BPIFB4 is associated with exceptional longevity: four single-nucleotide polymorphisms distinguish the wild-type form from a longevity-associated variant conferring positive effects on blood pressure. The effect of a rare variant (RV; allele frequency, 4%) on blood pressure is unknown. Here, we show that overexpression of RV-BPIFB4 in ex-vivo mouse vessels impairs phosphorylation of endothelial nitric oxide synthase (eNOS), blunting acetylcholine-evoked vasorelaxation; in vivo, virally mediated overexpression of RV-BPIFB4 increases blood pressure, an action absent in eNOS-deficient mice. In humans, we found RV carriers to have increased diastolic blood pressure, a finding that was more marked in subjects on anti-hypertensive medication; moreover, recombinant RV-BPIFB4 protein impaired eNOS function in ex-vivo human vessels. Thus, RV-BPIFB4 acts directly on blood pressure homeostasis and may represent a novel biomarker of vascular dysfunction and hypertension.

A Decrease of Brain MicroRNA-122 Level Is an Early Marker of Cerebrovascular Disease in the Stroke-Prone Spontaneously Hypertensive Rat

Based on preliminary evidence that highlights microRNA-122 as a contributing factor to stroke pathogenesis, we aimed at assessing its expression level, along with the presence of early signs of cerebrovascular disease, in the brain of stroke-prone spontaneously hypertensive rat (SHRSP), a suitable model of human disease that accelerates stroke occurrence under a high sodium/low potassium (Japanese-style) diet (JD). After one month of JD, before stroke occurrence, brain microRNA-122 level was significantly decreased in SHRSP as compared to the stroke-resistant SHR (SHRSR). At this time, levels of markers of oxidative stress and inflammation, as well as of endothelial integrity and function, apoptosis and necrosis were differently modulated in the brains of JD-fed SHRSP as compared to SHRSR, pointing to a significant activation of all deleterious mechanisms underlying subsequent stroke development in SHRSP. We also showed that miR-122 improved survival of rat endothelial cerebral cells upon stress stimuli (excess NaCl, hydrogen peroxide). Our data suggest that a decrease of brain microRNA-122 level is deleterious and can be considered as an early marker of stroke in the SHRSP. Understanding the mechanisms by which microRNA-122 protects vascular cells from stress stimuli may provide a useful approach to improve preventive and treatment strategies against stroke.

Methamphetamine increases prion protein and induces dopamine-dependent expression of protease resistant PrPsc

The cellular prion protein (PrPc) is physiologically expressed within selective brain areas of mammals. Alterations in the secondary structure of this protein lead to scrapie-like prion protein (PrPsc), which precipitates in the cell. PrPsc has been detected in infectious, inherited or sporadic neurodegenerative disorders. Prion protein metabolism is dependent on autophagy and ubiquitin proteasome. Despite not being fully elucidated, the physiological role of prion protein relates to chaperones which rescue cells under stressful conditions.Methamphetamine (METH) is a widely abused drug which produces oxidative stress in various brain areas causing mitochondrial alterations and protein misfolding. These effects produce a compensatory increase of chaperones while clogging cell clearing pathways. In the present study, we explored whether METH administration modifies the amount of PrPc. Since high levels of PrPc when the clearing systems are clogged may lead to its misfolding into PrPsc, we further tested whether METH exposure triggers the appearance of PrPsc. We analysed the effects of METH and dopamine administration in PC12 and striatal cells by using SDS-PAGE Coomassie blue, immune- histochemistry and immune-gold electron microscopy. To analyze whether METH administration produces PrPsc aggregates we used antibodies directed against PrP following exposure to proteinase K or sarkosyl which digest folded PrPc but misfolded PrPsc. We fond that METH triggers PrPsc aggregates in DA-containing cells while METH is not effective in primary striatal neurons which do not produce DA. In the latter cells exogenous DA is needed to trigger PrPsc accumulation similarly to what happens in DA containing cells under the effects of METH. The present findings, while fostering novel molecular mechanisms involving prion proteins, indicate that, cell pathology similar to prion disorders can be mimicked via a DA-dependent mechanism by a drug of abuse.

The emerging role of m-TOR up-regulation in brain Astrocytoma

The present manuscript is an overview of various effects of mTOR up-regulation in astrocytoma with an emphasis on its deleterious effects on the proliferation of Glioblastoma Multiforme. The manuscript reports consistent evidence indicating the occurrence of mTOR up-regulation both in experimental and human astrocytoma. The grading of human astrocytoma is discussed in relationship with mTOR up-regulation. In the second part of the manuscript, the biochemical pathways under the influence of mTOR are translated to cell phenotypes which are generated by mTOR up-regulation and reverted by its inhibition. A special section is dedicated to the prominent role of autophagy in mediating the effects of mTOR in glioblastoma. In detail, autophagy inhibition produced by mTOR up-regulation determines the fate of cancer stem cells. On the other hand, biochemical findings disclose the remarkable effects of autophagy activators as powerful inducers of cell differentiation with a strong prevalence towards neuronal phenotypes. Thus, mTOR modulation acts on the neurobiology of glioblastoma just like it operates in vivo at the level of brain stem cell niches by altering autophagy-dependent cell differentiation. In the light of such a critical role of autophagy we analyzed the ubiquitin proteasome system. The merging between autophagy and proteasome generates a novel organelle, named autophagoproteasome which is strongly induced by mTOR inhibitors in glioblastoma cells. Remarkably, when mTOR is maximally inhibited the proteasome component selectively moves within autophagy vacuoles, thus making the proteasome activity dependent on the entry within autophagy compartment.

A Focus on the Beneficial Effects of Alpha Synuclein and a Re-Appraisal of Synucleinopathies

Alpha synuclein (α-syn) belongs to a class of proteins which are commonly considered to play a detrimental role in neuronal survival. This assumption is based on the occurrence of a severe neuronal degeneration in patients carrying a multiplication of the α-syn gene (SNCA) and in a variety of experi-mental models, where overexpression of α-syn leads to cell death and neurological impairment. In these conditions, a higher amount of normally structured α-syn produces a damage, which is even worse com-pared with that produced by α-syn owning an abnormal structure (as occurring following point gene muta-tions). In line with this, knocking out the expression of α-syn is reported to protect from specific neurotox-ins such as 1-methyl, 4-phenyl 1,2,3,6-tetrahydropyridine (MPTP). In the present review we briefly dis-cuss these well-known detrimental effects but we focus on findings showing that, in specific conditions α-syn is beneficial for cell survival. This occurs during methamphetamine intoxication which is counteracted by endogenous α-syn. Similarly, the dysfunction of the chaperone cysteine-string protein-alpha leads to cell pathology which is counteracted by over-expressing α-syn. In line with this, an increased expression of α-syn protects against oxidative damage produced by dopamine. Remarkably, when the lack of α-syn is combined with a depletion of β- and γ- synucleins, alterations in brain structure and function occur. This review tries to balance the evidence showing a beneficial effect with the bulk of data reporting a detri-mental effect of endogenous α-syn. The specific role of α-syn as a chaperone protein is discussed to ex-plain such a dual effect.

Anti-inflammatory effect of a novel locally acting A2A receptor agonist in a rat model of oxazolone-induced colitis

Adenosine represents a powerful modulating factor, which has been shown to orchestrate the scope, duration, and remission of the inflammatory response through the activation of four specific receptors, classified as A1, A2A, A2B, and A3, all being widely expressed in a variety of immune cells. Several selective A2A receptor agonists have displayed anti-inflammatory effects, through the suppression of IL-12, TNF, and IFN-γ production by monocytes and lymphocytes, in the setting of chronic intestinal inflammation. However, the therapeutic application of A2A receptor agonists remains hindered by the risk of serious cardiovascular adverse effects arising from the wide systemic distribution of A2A receptors. The present study focused on evaluating the anti-inflammatory effects of the novel poorly absorbed A2A receptor agonist PSB-0777 in a rat model of oxazolone-induced colitis as well as to evaluate its cardiovascular adverse effects, paying particular attention to the onset of hypotension, one of the main adverse effects associated with the systemic pharmacological activation of A2A receptors. Colitis was associated with decreased body weight, an enhanced microscopic damage score and increased levels of colonic myeloperoxidase (MPO). PSB-0777, but not dexamethasone, improved body weight. PSB-0777 and dexamethasone ameliorated microscopic indexes of inflammation and reduced MPO levels. The beneficial effects of PSB-0777 on inflammatory parameters were prevented by the pharmacological blockade of A2A receptors. No adverse cardiovascular events were observed upon PSB-0777 administration. The novel A2A receptor agonist PSB-0777 could represent the base for the development of innovative pharmacological entities able to act in an event-specific and site-specific manner.

mTOR-Related Brain Dysfunctions in Neuropsychiatric Disorders

The mammalian target of rapamycin (mTOR) is an ubiquitously expressed serine-threonine kinase, which senses and integrates several intracellular and environmental cues to orchestrate major processes such as cell growth and metabolism. Altered mTOR signalling is associated with brain malformation and neurological disorders. Emerging evidence indicates that even subtle defects in the mTOR pathway may produce severe effects, which are evident as neurological and psychiatric disorders. On the other hand, administration of mTOR inhibitors may be beneficial for a variety of neuropsychiatric alterations encompassing neurodegeneration, brain tumors, brain ischemia, epilepsy, autism, mood disorders, drugs of abuse, and schizophrenia. mTOR has been widely implicated in synaptic plasticity and autophagy activation. This review addresses the role of mTOR-dependent autophagy dysfunction in a variety of neuropsychiatric disorders, to focus mainly on psychiatric syndromes including schizophrenia and drug addiction. For instance, amphetamines-induced addiction fairly overlaps with some neuropsychiatric disorders including neurodegeneration and schizophrenia. For this reason, in the present review, a special emphasis is placed on the role of mTOR on methamphetamine-induced brain alterations.

Systematic Morphometry of Catecholamine Nuclei in the Brainstem

Catecholamine nuclei within the brainstem reticular formation (RF) play a pivotal role in a variety of brain functions. However, a systematic characterization of these nuclei in the very same experimental conditions is missing so far. Tyrosine hydroxylase (TH) immune-positive cells of the brainstem correspond to dopamine (DA)-, norepinephrine (NE)-, and epinephrine (E)-containing cells. Here, we report a systematic count of TH-positive neurons in the RF of the mouse brainstem by using stereological morphometry. All these nuclei were analyzed for anatomical localization, rostro-caudal extension, volume, neuron number, neuron density, and mean neuronal area for each nucleus. The present data apart from inherent informative value wish to represent a reference for neuronal mapping in those studies investigating the functional anatomy of the brainstem RF. These include: the sleep-wake cycle, movement control, muscle tone modulation, mood control, novelty orienting stimuli, attention, archaic responses to internal and external stressful stimuli, anxiety, breathing, blood pressure, and innumerable activities modulated by the archaic iso-dendritic hard core of the brainstem RF. Most TH-immune-positive cells fill the lateral part of the RF, which indeed possesses a high catecholamine content. A few nuclei are medial, although conventional nosography considers all these nuclei as part of the lateral column of the RF. Despite the key role of these nuclei in psychiatric and neurological disorders, only a few of them aspired a great attention in biomedical investigation, while most of them remain largely obscure although intense research is currently in progress. A simultaneous description of all these nuclei is not simply key to comprehend the variety of brainstem catecholamine reticular neurons, but probably represents an intrinsically key base for understanding brain physiology and physiopathology.