Methamphetamine persistently increases alpha-synuclein and suppresses gene promoter methylation within striatal neurons

Abstract Methamphetamine (Meth) produces a variety of epigenetic effects in the brain, which are seminal to establish long-lasting alterations in neuronal activity. However, most epigenetic changes were described by measuring the rough amount of either histone acetylation and methylation or direct DNA methylation, without focusing on a specific DNA sequence. This point is key to comprehend Meth-induced phenotypic changes, brain plasticity, addiction and neurodegeneration. In this research paper we analyze the persistence of Meth-induced striatal synucleinopathy at a prolonged time interval of Meth withdrawal. At the same time, Meth-induced alterations, specifically within alpha-synuclein gene (SNCA) or its promoter, were evaluated. We found that exposure to high and/or prolonged doses of Meth, apart from producing nigro-striatal toxicity, determines a long-lasting increase in striatal alpha-synuclein levels. This is consistent along immune-blotting, immune-histochemistry, and electron microscopy. This was neither associated with an increase of SNCA copy number nor with alterations within SNCA sequence. However, we documented persistently demethylation within SNCA promoter, which matches the increase in alpha-synuclein protein. The amount of the native protein, which was measured stoichiometrically within striatal neurons, surpasses the increase reported following SNCA multiplications. Demethylation was remarkable (ten-fold of controls) and steady, even at prolonged time intervals being tested so far (up to 21 days of Meth withdrawal). Similarly alpha-synuclein protein assayed stoichiometrically steadily increased roughly ten-fold of controls. Meth-induced increase of alpha-synuclein was also described within limbic areas. These findings are discussed in the light of Meth-induced epigenetic changes, Meth-induced phenotype alterations, and Meth-induced neurodegeneration

Persistent paradoxical eff ects on striatal and limbic a-synuclein and tyrosine hydroxylase following methamphetamine withdrawal

Methamphetamine (METH) produces a variety of epigenetic eff ects in the brain, which are seminal to establish long-lasting alterations in neuronal activity. A number of studies were car ried out aimed at rough assessment of the amount of either histone acetylation and methyla tion or direct DNA methylation, without a selective analysis of specifi c genes. In the present study we wish to assess whether METH-induced epigenetic alterations may specifi cally engage the expression of a-synuclein, which is a key protein in neurodegeneration and synaptic plastic ity. In this way, a potential long-term alteration of brain circuitries may produce a variation in the threshold for neurotoxicity, sensitization, addiction and neurodegeneration. Thus, the occur rence of long-term changes in the expression of the protein were analyzed in parallel with per sistent changes in a specifi c marker of integrity of meso-striatal/meso-limbic pathway, which is the expression of tyrosine hydroxylase (TH) both in the mesencephalon and within dorsal striatum. The integrity of dopamine (DA) projection was assessed at the level of the olfactory tubercle, the nucleus accumbens and fundus striati. Prolonged exposure to small doses of METH, produces nigro-striatal toxicity, when assessed at short time intervals following prolonged exposure. However, at prolonged time intervals a paradoxical increase progressively occurred in TH immunostaining within limbic regions. Such an increase exceeds at large the amount of TH expressed in controls. This occurs concomitantly with an overexpression of the primary transcript as well as the protein alpha synuclein within the same brain regions and dorsal striatum. This increase is persistent at prolonged time inter val of METH withdrawal.The increase in the primary a-synuclein transcript is due to hypomethylation of specifi c CPG islands placed in the SNCA gene promoter which ranged roughly ten-fold of controls, it was steady, and it persisted at least 21 days following METH withdrawal. Thus, such an appar ent synucleinopathy induced by METH indeed was associated with increased mesolimbic DA innervation, which equally surpasses several folds the amount which was measured in controls and persists at least for three weeks. The increase in SNCA is not associated with an increase of SNCA copy number. Nonetheless, the amount of the native protein, which is detected by ultra structural stoichiometry, exceeds the increase reported following genetic SNCA multiplications (ten-fold of controls). These fi ndings are discussed in the light of METH-induced phenotype changes which accompany toxicity, sensitization, addiction and neurodegeneration

Chronic MPTP in Mice Damage-specific Neuronal Phenotypes within Dorsal Laminae of the Spinal Cord

The neurotoxin 1-methyl, 4-phenyl, 1, 2, 3, 6-tetrahydropiridine (MPTP) is widely used to produce experimental parkinsonism. Such a disease is characterized by neuronal damage in multiple regions beyond the nigrostriatal pathway including the spinal cord. The neurotoxin MPTP damages spinal motor neurons. So far, in Parkinson’s disease (PD) patients alpha-synuclein aggregates are described in the dorsal horn of the spinal cord. Nonetheless, no experimental investigation was carried out to document whether MPTP affects the sensory compartment of the spinal cord. Thus, in the present study, we investigated whether chronic exposure to small doses of MPTP (5 mg/kg/X2, daily, for 21 days) produces any pathological effect within dorsal spinal cord. This mild neurotoxic protocol produces a damage only to nigrostriatal dopamine (DA) axon terminals with no decrease in DA nigral neurons assessed by quantitative stereology. In these experimental conditions we documented a decrease in enkephalin-, calretinin-, calbindin D28K-, and parvalbumin-positive neurons within lamina I and II and the outer lamina III. Met-Enkephalin and substance P positive fibers are reduced in laminae I and II of chronically MPTP-treated mice. In contrast, as reported in PD patients, alpha-synuclein is markedly increased within spared neurons and fibers of lamina I and II after MPTP exposure. This is the first evidence that experimental parkinsonism produces the loss of specific neurons of the dorsal spinal cord, which are likely to be involved in sensory transmission and in pain modulation providing an experimental correlate for sensory and pain alterations in PD

Quantitative ultrastructural morphometry and gene expression of mTOR-related mitochondriogenesis within glioblastoma cells

In glioblastoma (GBM) cells, an impairment of mitochondrial activity along with autophagy suppression occurs. Autophagy suppression in GBM promotes stemness, invasion, and poor prognosis. The autophagy deficit seems to be due, at least in part, to an abnormal up-regulation of the mammalian target of rapamycin (mTOR), which may be counteracted by pharmacological mTORC1 inhibition. Since autophagy activation is tightly bound to increased mitochondriogenesis, a defect in the synthesis of novel mitochondria is expected to occur in GBM cells. In an effort to measure a baseline deficit in mitochondria and promote mitochondriogenesis, the present study used two different GBM cell lines, both featuring mTOR hyperactivity. mTORC1 inhibition increases the expression of genes and proteins related to autophagy, mitophagy, and mitochondriogenesis. Autophagy activation was counted by RT-PCR of autophagy genes, LC3-immune-fluorescent puncta and immune-gold, as well as specific mitophagy-dependent BNIP3 stoichiometric increase in situ, within mitochondria. The activation of autophagy-related molecules and organelles after rapamycin exposure occurs concomitantly with progression of autophagosomes towards lysosomes. Remarkably, mitochondrial biogenesis and plasticity (increased mitochondrial number, integrity, and density as well as decreased mitochondrial area) was long-lasting for weeks following rapamycin withdrawal

An attempt to dissect a peripheral marker based on cell pathology in Parkinson's disease

Peripheral markers in Parkinson’s disease (PD) represent a hot issue to provide early diagnosis and assess disease progression. The gold standard marker of PD should feature the same reliability as the pathogenic alteration, which produces the disease itself. PD is foremost a movement disorder produced by a loss of nigrostriatal dopamine innervation, in which striatal dopamine terminals are always markedly reduced in PD patients to an extent, which never overlaps with controls. Similarly, a reliable marker of PD should possess such a non-overlapping feature when compared with controls. In the present study, we provide a novel pathological hallmark, the autophagosome, which in each PD patient was always suppressed compared with each control subject. Autophagosomes were counted as microtubule-associated proteins 1A/1B light chain 3B (LC3)-positive vacuoles at ultrastructural morphometry within peripheral (blood) blood mononuclear cells (PBMC). This also provides the gold standard to assess the autophagy status. Since autophagy may play a role in the pathogenesis of PD, autophagosomes may be a disease marker, while participating in the biology of the disease. Stoichiometric measurement of α-synuclein despite significantly increased in PD patients, overlapped between PD and control patients. Although the study need to be validated in large populations, the number of autophagy vacuoles is neither related with therapy (the amount was similarly suppressed in a few de novo patients), nor the age in PD or controls

Audio Books with Struggling Readers at the Elementary School Level

In a Title I school located in a southeastern state, 60% of 3rd grade students are reading below grade level. The state\u27s new reading initiative ties grade promotion to 3rd grade students reading on grade level. At the study site, administrators identified audio books as a possibly helpful reading tool. Vygotsky\u27s zone of proximal development theory, which holds that learners can learn new skills more readily with guided assistance, framed this study. The purpose of this quantitative, comparative design study was to explore the associations between the use of audio books and the reading levels of 3rd grade struggling readers. Research questions were used to compare the reading levels of struggling readers who use audio books with the reading levels of: (a) struggling readers reading silently, (b) at or above grade level readers who read with audio books, and (c) at or above grade level readers who read silently. Two 3rd grade classes were selected, with 25 students using audio books and 25 students reading silently, to participate in this project. Scores from the AR and from the pre- and posttest STAR assessments over a 9-week period were analyzed and compared using an independent samples t test to explore associations between the use of audio books and the comprehension and reading levels of the participants. Analysis of the results showed that the use of audiobooks was not significantly related to increased reading or comprehension levels for struggling readers. Significant improvements in reading comprehension were shown for students reading at or above grade level that read silently or used audio books. Based on the findings, a professional development project for teachers providing research-supported reading strategy instruction was developed. The findings may lead to improvements in instructional practices by encouraging the use of research-based reading strategies, which could promote positive social change by supporting greater academic success for elementary students through improved reading comprehension

Rapamycin promotes differentiation increasing βIII-tubulin, NeuN, and NeuroD while suppressing nestin expression in glioblastoma cells

Glioblastoma cells feature mammalian target of rapamycin (mTOR) up-regulation which relates to a variety of effects such as: lower survival, higher infiltration, high stemness and radio- and chemo-resistance. Recently, it was demonstrated that mTOR may produce a gene shift leading to altered protein expression. Therefore, in the present study we administered different doses of the mTOR inhibitor rapamycin to explore whether the transcription of specific genes are modified. By using a variety of methods we demonstrate that rapamycin stimulates gene transcription related to neuronal differentiation while inhibiting stemness related genes such as nestin. In these experimental conditions, cell phenotype shifts towards a pyramidal neuron-like shape owing long branches. Rapamycin suppressed cell migration when exposed to fetal bovine serum (FBS) while increasing the cell adhesion protein phospho-FAK (pFAK). The present study improves our awareness of basic mechanisms which relate mTOR activity to the biology of glioblastoma cells. These findings apply to a variety of effects which can be induced by mTOR regulation in the brain. In fact, the ability to promote neuronal differentiation might be viewed as a novel therapeutic pathway to approach neuronal regeneration

A novel POLR3A genotype leads to leukodystrophy type-7 in two siblings with unusually late age of onset

Background: Leukodystrophies are familial heterogeneous disorders primarily affecting the white matter, which are defined as hypomyelinating or demyelinating based on disease severity as assessed at MRI. Recently, a group of clinically overlapping hypomyelinating leukodystrophies (HL) has been associated with mutations in RNA polymerase III enzymes (Pol III) subunits. Case presentation: In this manuscript, we describe two Italian siblings carrying a novel POLR3A genotype. MRI imaging, genetic analysis, and clinical data led to diagnosing HL type 7. The female sibling, at the age of 34, is tetra-paretic and suffers from severe cognitive regression. She had a disease onset at the age of 19, characterized by slow and progressive cognitive impairment associated with gait disturbances and amenorrhea. The male sibling was diagnosed during an MRI carried out for cephalalgia at the age of 41. After 5 years, he developed mild cognitive impairment, dystonia with 4-limb hypotonia, and moderate dysmetria with balance and gait impairment. Conclusions: The present study provides the first evidence of unusually late age of onset in HL, describing two siblings with a novel POLR3A genotype which showed the first symptoms at the age of 41 and 19, respectively. This provides a powerful insight into clinical heterogeneity and genotype-phenotype correlation in POLR3A related HL

A Large Family with p.Arg554His Mutation in ABCD1: Clinical Features and Genotype/Phenotype Correlation in Female Carriers

X-linked adrenoleukodystrophy (X-ALD, OMIM #300100) is the most common peroxisomal disorder clinically characterized by two main phenotypes: adrenomyeloneuropathy (AMN) and the cerebral demyelinating form of X-ALD (cerebral ALD). The disease is caused by defects in the gene for the adenosine triphosphate (ATP)-binding cassette protein, subfamily D (ABCD1) that encodes the peroxisomal transporter of very-long-chain fatty acids (VLCFAs). The defective function of ABCD1 protein prevents β-oxidation of VLCFAs, which thus accumulate in tissues and plasma, to represent the hallmark of the disease. As in many X-linked diseases, it has been routinely expected that female carriers are asymptomatic. Nonetheless, recent findings indicate that most ABCD1 female carriers become symptomatic, with a motor disability that typically appears between the fourth and fifth decade. In this paper, we report a large family in which affected males died during the first decade, while affected females develop, during the fourth decade, progressive lower limb weakness with spastic or ataxic-spastic gait, tetra-hyperreflexia with sensory alterations. Clinical and genetic evaluations were performed in nine subjects, eight females (five affected and three healthy) and one healthy male. All affected females were carriers of the c.1661G>A (p.Arg554His, rs201568579) mutation. This study strengthens the relevance of clinical symptoms in female carriers of ABCD1 mutations, which leads to a better understanding of the role of the genetic background and the genotype-phenotype correlation. This indicates the relevance to include ABCD1 genes in genetic panels for gait disturbance in women

Decipher non-canonical SPAST splicing mutations with the help of functional assays in patients affected by spastic paraplegia 4 (SPG4)

Flowchart showing the molecular approach used to decipher the non-canonical splicing mutations