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Neurodegenerative disorders are typically featured by the occurrence of neuronal inclusions. In the case of Parkinson's disease (PD) these correspond to Lewy bodies (LBs), which are routinely defined as proteinaceous inclusions composed of alpha-synuclein (alpha-syn). In turn, alpha-syn is considered to be the key protein in producing PD and fostering its progression. Recent studies challenged such a concept and emphasized the occurrence of other proteins such as p62 and poly-ubiquitin (Poly-ub) in the composition of LBs, which are also composed of large amounts of tubulo-vesicular structures. All these components, which accumulate within the cytosol of affected neurons in PD, may be the consequence of a dysfunction of major clearing pathways. In fact, autophagy-related systems are constantly impaired in inherited PD and genetic models of PD. The present study was designed to validate whether a pharmacological inhibition of autophagy within catecholamine cells produces cell damage and accumulation of specific proteins and tubulo-vesicular structures. The stoichiometry counts of single proteins, which accumulate within catecholamine neurons was carried out along with the area of tubulo-vesicular structures. In these experimental conditions p62 and Poly-ub accumulation exceeded at large the amounts of alpha-syn. In those areas where Poly-ub and p62 were highly expressed, tubulo-vesicular structures were highly represented compared with surrounding cytosol. The present study confirms new vistas about LBs composition and lends substance to the scenario that autophagy inhibition rather than a single protein dysfunction as key determinant of PD., Competing Interests: Declarations. Conflict of interest: The authors have no competing interests to declare that are relevant to the content of this article., (© 2024. The Author(s).)

Methamphetamine (METH) produces a cytopathology, which is rather specific within catecholamine neurons both in vitro and ex vivo, in animal models and chronic METH abusers. This led some authors to postulate a sort of parallelism between METH cytopathology and cell damage in Parkinson's disease (PD). In fact, METH increases and aggregates alpha-syn proto-fibrils along with producing spreading of alpha-syn. Although alpha-syn is considered to be the major component of aggregates and inclusions developing within diseased catecholamine neurons including classic Lewy body (LB), at present, no study provided a quantitative assessment of this protein in situ, neither following METH nor in LB occurring in PD. Similarly, no study addressed the quantitative comparison between occurrence of alpha-syn and other key proteins and no investigation measured the protein compared with non-protein structure within catecholamine cytopathology. Therefore, the present study addresses these issues using an oversimplified model consisting of a catecholamine cell line where the novel approach of combined light and electron microscopy (CLEM) was used measuring the amount of alpha-syn, which is lower compared with p62 or poly-ubiquitin within pathological cell domains. The scenario provided by electron microscopy reveals unexpected findings, which are similar to those recently described in the pathology of PD featuring packing of autophagosome-like vesicles and key proteins shuttling autophagy substrates. Remarkably, small seed-like areas, densely packed with p62 molecules attached to poly-ubiquitin within wide vesicular domains occurred. The present data shed new light about quantitative morphometry of catecholamine cell damage in PD and within the addicted brain., (© 2024. The Author(s).)

Locus Coeruleus (LC) is the main noradrenergic nucleus of the brain, which is involved in many physiological functions including cognition; its impairment may be crucial in the neurobiology of a variety of brain diseases. Locus Coeruleus-Magnetic Resonance Imaging (LC-MRI) allows to identify in vivo LC in humans. Thus, a variety of research teams have been using LC-MRI to estimate LC integrity in normal aging and in patients affected by neurodegenerative disorders, where LC integrity my work as a biomarker. A number of variations between LC-MRI studies exist, concerning post-acquisition analysis and whether this had been performed within MRI native space or in ad hoc-built MRI template space. Moreover, the reproducibility and reliability of this tool is still to be explored. Therefore, in the present study, we analyzed a group of neurologically healthy, cognitively intact elderly subjects, using both a native space- and a template space-based LC-MRI analysis. We found a good inter-method agreement, particularly considering the LC Contrast Ratio. The template space-based approach provided a higher spatial resolution, lower operator-dependency, and allowed the analysis of LC topography. Our ad hoc-developed LC template showed LC morphological data that were in line with templates published very recently. Remarkably, present data significantly overlapped with a recently published LC "metaMask", that had been obtained by averaging the results of a variety of previous LC-MRI studies. Thus, such a template space-based approach may pave the way to a standardized LC-MRI analysis and to be used in future clinic-anatomical correlations., (© 2022. The Author(s).)

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., (© 2021. The Author(s).)

Spinal muscular atrophy (SMA) is a heritable, autosomal recessive neuromuscular disorder characterized by a loss of the survival of motor neurons (SMN) protein, which leads to degeneration of lower motor neurons, and muscle atrophy. Despite SMA being nosographically classified as a motor neuron disease, recent advances indicate that peripheral alterations at the level of the neuromuscular junction (NMJ), involving the muscle, and axons of the sensory-motor system, occur early, and may even precede motor neuron loss. In the present study, we used a mouse model of slow progressive (type III) SMA, whereby the absence of the mouse SMN protein is compensated by the expression of two human genes (heterozygous SMN1A2G, and SMN2). This leads to late disease onset and prolonged survival, which allows for dissecting slow degenerative steps operating early in SMA pathogenesis. In this purely morphological study carried out at transmission electron microscopy, we extend the examination of motor neurons and proximal axons towards peripheral components, including distal axons, muscle fibers, and also muscle spindles. We document remarkable ultrastructural alterations being consistent with early peripheral denervation in SMA, which may shift the ultimate anatomical target in neuromuscular disease from the spinal cord towards the muscle. This concerns mostly mitochondrial alterations within distal axons and muscle, which are quantified here through ultrastructural morphometry. The present study is expected to provide a deeper knowledge of early pathogenic mechanisms in SMA.

The hypothalamus and Locus Coeruleus (LC) share a variety of functions, as both of them take part in the regulation of the sleep/wake cycle and in the modulation of autonomic and homeostatic activities. Such a functional interplay takes place due to the dense and complex anatomical connections linking the two brain structures. In Alzheimer's disease (AD), the occurrence of endocrine, autonomic and sleep disturbances have been associated with the disruption of the hypothalamic network; at the same time, in this disease, the occurrence of LC degeneration is receiving growing attention for the potential roles it may have both from a pathophysiological and pathogenetic point of view. In this review, we summarize the current knowledge on the anatomical and functional connections between the LC and hypothalamus, to better understand whether the impairment of the former may be responsible for the pathological involvement of the latter, and whether the disruption of their interplay may concur to the pathophysiology of AD. Although only a few papers specifically explored this topic, intriguingly, some pre-clinical and post-mortem human studies showed that aberrant protein spreading and neuroinflammation may cause hypothalamus degeneration and that these pathological features may be linked to LC impairment. Moreover, experimental studies in rodents showed that LC plays a relevant role in modulating the hypothalamic sleep/wake cycle regulation or neuroendocrine and systemic hormones; in line with this, the degeneration of LC itself may partly explain the occurrence of hypothalamic-related symptoms in AD.

Autophagy is a degradative process playing an important role in removing misfolded or aggregated proteins, clearing damaged organelles, such as mitochondria and endoplasmic reticulum, as well as eliminating intracellular pathogens. The autophagic process is important for balancing sources of energy at critical developmental stages and in response to nutrient stress. Recently, autophagy has been involved in the pathophysiology of neurodegenerative diseases although its beneficial (pro-survival) or detrimental (pro-death) role remains controversial. In the present review, we discuss the role of autophagy following intoxication with trimethyltin (TMT), an organotin compound that induces severe hippocampal neurodegeneration associated with astrocyte and microglia activation. TMT is considered a useful tool to study the molecular mechanisms occurring in human neurodegenerative diseases such as Alzheimer's disease and temporal lobe epilepsy. This is also relevant in the field of environmental safety, since organotin compounds are used as heat stabilizers in polyvinyl chloride polymers, industrial and agricultural biocides, and as industrial chemical catalysts.

Protease-activated receptor 1 (PAR1) is a cell surface receptor, which belongs to a family of G protein-coupled receptors and signals in response to multiple extracellular proteases. PAR1 is widely distributed in mammalian cells and tissues, including human glial cells. Within this context, PAR1 may participate to various activities promoted by glial cells. In fact, glia does not represent merely a glue in the nervous system but affects significantly various neuronal functions and activities being also significantly involved in the pathophysiology of various nervous system disorders. In this review, we summarize the current understanding of PAR1 expression and functions within glial cells both in the central and peripheral nervous system.

Recent evidence suggests that autophagy alterations are present in a variety of neurological disorders. These range from neurodegenerative diseases to acute neurological insults. Thus, despite a role of autophagy was investigated in a variety of neurological diseases, only recently these studies included epilepsy. This was fostered by the evidence that rapamycin, a powerful autophagy inducer, strongly modulates a variety of seizure models and epilepsies. These findings were originally interpreted as the results of the inhibition exerted by rapamycin on the molecular complex named "mammalian Target of Rapamycin" (mTOR). Recently, an increasing number of papers demonstrated that mTOR inhibition produces a strong activation of the autophagy machinery. In this way, it is now increasingly recognized that what was once defined as mTORpathy in epileptogenesis may be partially explained by abnormalities in the autophagy machinery. The present review features a brief introductory statement about the autophagy machinery and discusses the involvement of autophagy in seizures and epilepsies. An emphasis is posed on evidence addressing both pros and cons making it sometime puzzling and sometime evident, the role of autophagy in the epileptic brain.

We induced brief secondarily generalized seizures of limbic origin in Sprague-Dawley rats by bicuculline microinfusion into the anterior piriform cortex. After 1 h or 5 days we performed comet assay, a sensitive marker for DNA damage, within entorhinal cortex, hippocampus (limbic areas recruited by seizure spreading) and striatum (which is not recruited). DNA damage occurred selectively in the ipsilateral entorhinal cortex and hippocampus at 1 h, but not at 5 days. These data shed new light on molecular genetics as a marker during limbic seizures, the most common in epileptic patients.

Trimethyltin (TMT) is a triorganotin compound which determines neurodegeneration of specific brain areas particularly damaging the limbic system. Earlier ultrastructural studies indicated the formation of autophagic vacuoles in neurons after TMT intoxication. However, no evaluation has been attempted to determine the role of the autophagic pathway in TMT neurotoxicity. To assess the contribution of autophagy to TMT-induced neuronal cell death, we checked the vulnerability of neuronal cultures to TMT after activation or inhibition of autophagy. Our results show that autophagy inhibitors (3-methyladenine and L-asparagine) greatly enhanced TMT neurotoxicity. Conversely, known activators of autophagy, such as lithium and rapamycin, displayed neuroprotection against this toxic compound. Due to its diverse targets, the action of lithium was complex. When lithium was administered according to a chronic treatment protocol (6 days pretreatment) it was able to rescue both hippocampal and cortical neurons from TMT (or from glutamate toxicity used as reference). This effect was accompanied by an increased phosphorylation of glycogen synthase kinase 3 which is a known target for lithium neuroprotection. If the pre-incubation time was reduced to 2 h (acute treatment protocol), lithium was still able to counteract TMT toxicity in hippocampal but not in cortical neurons. The neuroprotective effect of lithium acutely administered against TMT in hippocampal neurons can be completely reverted by an excess of inositol and is possibly related to the inactivation of inositol monophosphatase, a key regulator of autophagy. These data indicate that TMT neurotoxicity can be dramatically modified, at least in vitro, by lithium addition which seems to act through different mechanisms if acutely or chronically administered.

Huntington's Disease (HD) is caused by trinucleotide CAG repeat expansion >36 in huntingtin (htt), a protein with several documented functions. The elongated polyglutamine (polyQ) stretch in the N-terminal region of htt leads to dysfunctional and degenerative events in neurons and peripheral tissues. In this study, by extending the analysis to several caspase activities (i.e. caspase 2, 3, 6, 8 and 9), we describe genotype- and time- dependent caspase activity abnormalities, decreased cell viability and a large set of alterations in mitochondria morphology, in cultured blood cells from HD patients. Patients homozygous for CAG repeat mutations and heterozygous with high size mutations causing juvenile onset (JHD) presented significantly increased caspase 2, 3, 6, 8 and 9 activities, decreased cell viability and pronounced morphological abnormalities, compared with cells carrying low mutation size and controls. After cyanide treatment, all caspases increased their activities in homozygous and highly expanded heterozygous cells, caspase 8 and 9 increased also in those cells carrying low-size mutations, remarking their key role as 'caspase initiators' in HD. The remarkable ageing-dependent abnormalities in peripheral cells carrying particularly toxic mutations (i.e. homozygotes' and JHD's blood cells) points out the potential dependence of clinical HD development and progression on either mutated htt dosage or missing wild type htt. Peripheral tissues (i.e. blood cells) may theoretically represent an important tool for studying HD mechanisms and searching for new biomarkers, according to the patients' genotype.

We investigated the genotype-dependency of morphological abnormalities in peripheral cells from Huntington disease (HD) patients. Cell cultures derived from skin and muscle biopsies showed a different set of abnormalities depending on the genotype (i.e. heterozygous and homozygous for CAG mutations) and the tissue (i.e. fibroblasts and myoblasts). In general, homozygotes' cell lines showed massive ultrastructural damage of specific cell organelles compared with age matched control. These consist of vacuolization, deranged crests and matrix found within giant mitochondria. In addition, enlarged endoplasmic reticulum and the occurrence of numerous autophagic vacuoles, which were similar to those occurring in neurons within affected brain areas, were described. Despite a comparable dose-dependency on mitochondrial changes, this kind of alterations differ in fibroblasts compared with myoblasts. In fact, the internal mitochondrial structure was merely lost in myoblasts, while it shows pathological re-organization within fibroblasts, where altered crests appear as multilamellar circles. These data indicate that ultrastructural abnormalities from peripheral tissues of HD patients can be used as potential disease markers which are easier to get than autoptic brains. Moreover, the occurrence of ultrastructural cell pathology reminiscent of neuronal degeneration in HD, suggests the use of human peripheral cells as a tool to investigate the pathogenic cascade subsequent to huntingtin dysregulation.

Dopamine (DA) axons in the developing striatum cluster in discrete areas called "DA islands". During the third postnatal week, most DA islands are no-longer detectable and the DA innervation becomes uniform. In this study we explored the relationship between the pattern of DA innervation and the number of striatal tyrosine hydroxylase positive (TH+) cells during early postnatal development. By using dedicated stereology we found that the newborn striatum contains striatal TH+ cells, which cluster around newly sprouted DA axons. The number of these cells decreases when DA axons develop a full pattern of striatal innervation. This condition suggests a causal relationship between the amount of striatal DA innervation and the presence of striatal DA neurons. A better knowledge of the mechanisms regulating the ontogenesis of the nigrostriatal DA system may pave the way to strategies of neurorescue of the DA system.

The repeated finding of an apparent protective effect of cigarette smoking on the risk of Parkinson's disease is one of the few consistent results in the epidemiology of this disorder. Among the innumerous substances that originate from tobacco smoke, nicotine is by far the most widely studied, and the most likely candidate for a protective effect against neuronal degeneration in Parkinson's disease. Nicotine is a natural alkaloid that has considerable stimulatory effects on the central nervous system (CNS). Its effects on the CNS are mediated by the activation of neuronal heteromeric acetylcholine-gated ion channel receptors (nAChR, also termed nicotinic acetylcholine receptors). In the present study, we describe the neuroprotective effects of (-)nicotine in two animal models of parkinsonism: the diethyldithiocarbamate (DDC)-induced enhancement of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity in mice, and the methamphetamine-induced neurotoxicity in rats and mice. In parallel experiments, we found that (-)nicotine induces the basic fibroblast growth factor (FGF-2) and the brain-derived neurotrophic factor (BDNF) in rat striatum. As FGF-2 and BDNF have been reported to be neuroprotective for dopaminergic cells, our data indicate that the increase in neurotrophic factors is a possible mechanism by which (-)nicotine protects from experimental parkinsonisms. Moreover, they suggest that nAChR agonists could be of potential benefit in the progression of Parkinson's disease.