The neurodegenerative disease Friedreich's ataxia is caused by lower than normal levels of frataxin, an important protein involved in iron–sulfur (Fe-S) cluster biogenesis. An important step in designing strategies to treat this disease is to understand whether increasing the frataxin levels by gene therapy would simply be beneficial or detrimental, because previous studies, mostly based on animal models, have reported conflicting results. Here, we have exploited an inducible model, which we developed using the CRISPR/Cas9 methodology, to study the effects of frataxin overexpression in human cells and monitor how the system recovers after overexpression. Using new tools, which range from high-throughput microscopy to in cell infrared, we prove that overexpression of the frataxin gene affects the cellular metabolism. It also leads to a significant increase of oxidative stress and labile iron pool levels. These cellular alterations are similar to those observed when the gene is partly silenced, as occurs in Friedreich's ataxia patients. Our data suggest that the levels of frataxin must be tightly regulated and fine-tuned, with any imbalance leading to oxidative stress and toxicity.

Autophagy and endolysosomal trafficking are crucial in neuronal development, function and survival. These processes ensure efficient removal of misfolded aggregation-prone proteins and damaged organelles, such as dysfunctional mitochondria, thus allowing the maintenance of proper cellular homeostasis. Beside this, emerging evidence has pointed to their involvement in the regulation of the synaptic proteome needed to guarantee an efficient neurotransmitter release and synaptic plasticity. Along this line, an intimate interplay between the molecular machinery regulating synaptic vesicle endocytosis and synaptic autophagy is emerging, suggesting that synaptic quality control mechanisms need to be tightly coupled to neurosecretion to secure release accuracy. Defects in autophagy and endolysosomal pathway have been associated with neuronal dysfunction and extensively reported in Alzheimer’s, Parkinson’s, Huntington’s and amyotrophic lateral sclerosis among other neurodegenerative diseases, with common features and emerging genetic bases. In this review, we focus on the multiple roles of autophagy and endolysosomal system in neuronal homeostasis and highlight how their defects probably contribute to synaptic default and neurodegeneration in the above-mentioned diseases, discussing the most recent options explored for therapeutic interventions.

BACE1 and BACE2 are two closely related membrane-bound aspartic proteases. BACE1 is widely recognized as the neuronal β-secretase that cleaves the amyloid-β precursor protein, thus allowing the production of amyloid-β, i.e. the peptide that has been proposed to trigger the neurodegenerative process in Alzheimer's disease. BACE2 has ubiquitous expression and its physiological and pathological role is still unclear. In light of a possible role of glial cells in the accumulation of amyloid-β in brain, we have investigated the expression of these two enzymes in primary cultures of astrocytes. We show that astrocytes possess β-secretase activity and produce amyloid-β because of the activity of BACE2, but not BACE1, the expression of which is blocked at the translational level. Finally, our data demonstrate that changes in the astrocytic phenotype during neuroinflammation can produce both a negative as well as a positive modulation of β-secretase activity, also depending on the differential responsivity of the brain regions.

Neuropeptide Y (NPY) is a neuropeptide abundantly expressed in the mammalian central and peripheral nervous system. NPY is a pleiotropic molecule, which influences cell proliferation, cardiovascular and metabolic function, pain and neuronal excitability. In the central nervous system, NPY acts as a neuromodulator, affecting pathways that range from cellular (excitability, neurogenesis) to circuit level (food intake, stress response, pain perception). NPY has a broad repertoire of receptor subtypes, each activating specific signaling pathways in different tissues and cellular sub-regions. In the context of epilepsy, NPY is thought to act as an endogenous anticonvulsant that performs its action through Y2 and Y5 receptors. In fact, its overexpression in the brain with the aid of viral vectors can suppress seizures in animal models of epilepsy. Therefore, NPY-based gene therapy may represent a novel approach for the treatment of epilepsy patients, particularly for pharmaco-resistant and genetic forms of the disease. Nonetheless, considering all the aforementioned aspects of NPY signaling, the study of possible NPY applications as a therapeutic molecule is not devoid of critical aspects. The present review will summarize data related to NPY biology, focusing on its anti-epileptic effects, with a critical appraisal of key elements that could be exploited to improve the already existing NPY-based gene therapy approaches for epilepsy.

Neuronal physiology requires activity-driven protein translation, a process in which translation initiation factors are key players. We focus on eukaryotic initiation factor 4B (eIF4B), a regulator of protein translation, whose function in neurons is undetermined. We show that neuronal activity affects eIF4B phosphorylation and identify Ser504 as a phosphorylation site regulated by casein kinases and sensitive to the activation of metabotropic glutamate receptors. Ser504 phosphorylation increases eIF4B recruitment to the pre-initiation complex and influences eIF4B localization at synapses. Moreover, Ser504 phosphorylation modulates the translation of protein kinase Mζ. Therefore, by sensing synaptic activity, eIF4B could adjust translation to neuronal needs, promoting adaptive changes in synaptic plasticity. We also show that Ser504 phosphorylation is increased in vivo in a rat model of epilepsy during epileptogenesis i.e. when translation drives maladaptive synaptic changes. We propose eIF4B as a mediator between neuronal activity and translation, with relevance in the control of synaptic plasticity.

BackgroundCeruloplasmin is a ferroxidase expressed in the central nervous system both as soluble form in the cerebrospinal fluid (CSF) and as membrane-bound GPI-anchored isoform on astrocytes, where it plays a role in iron homeostasis and antioxidant defense. It has been proposed that ceruloplasmin is also able to activate microglial cells with ensuing nitric oxide (NO) production, thereby contributing to neuroinflammatory conditions. In light of the possible role of ceruloplasmin in neurodegenerative diseases, we were prompted to investigate how this protein could contribute to microglial activation in either its native form, as well as in its oxidized form, recently found generated in the CSF of patients with Parkinson’s and Alzheimer’s diseases.MethodsPrimary rat microglial-enriched cultures were treated with either ceruloplasmin or oxidized-ceruloplasmin, alone or in combination with lipopolysaccharide (LPS). Production of NO and expression of inducible nitric oxide synthase (iNOS) were evaluated by Griess assay and Western blot analysis, respectively. The productions of the pro-inflammatory cytokine IL-6 and the chemokine MIP-1α were assessed by quantitative RT-PCR and ELISA.ResultsRegardless of its oxidative status, ceruloplasmin by itself was not able to activate primary rat microglia. However, ceruloplasmin reinforced the LPS-induced microglial activation, promoting an increase of NO production, as well as the induction of IL-6 and MIP-1α. Interestingly, the ceruloplasmin-mediated effects were observed in the absence of an additional induction of iNOS expression. The evaluation of iNOS activity in primary glial cultures and in vitro suggested that the increased NO production induced by the combined LPS and ceruloplasmin treatment is mediated by a potentiation of the enzymatic activity.ConclusionsCeruloplasmin potentiates iNOS activity in microglial cells activated by a pro-inflammatory stimulus, without affecting iNOS expression levels. This action might be mediated by the activation of a yet unknown Cp receptor that triggers intracellular signaling that cross-talks with the response elicited by LPS or other pro-inflammatory stimuli. Therefore, ceruloplasmin might contribute to pathological conditions in the central nervous system by exacerbating neuroinflammation.

A turning point of research in Alzheimer’s disease was undoubtedly the discovery of BACE1, the amyloid-β precursor protein-cleaving enzyme that initiates the generation of amyloid-β, the peptide strongly suspected to be responsible for neuronal malfunction and death. Several research groups started a race to identify the best inhibitor of BACE1 activity. On the other hand, basic researchers are evaluating the changes in BACE1 expression and activity with the aim to better understand the pathogenetic process of the disease. Along this second line of research, in the last few years many important results have been reported in various experimental models, as well as in Alzheimer’s disease patients. As a consequence, new pathogenetic paradigms have been developed. We have reviewed these reports trying to highlight contrasting viewpoints, data awaiting final confirmation, and promising perspectives.

A key factor for developing gene therapy strategies for neurological disorders is the availability of suitable vectors. Currently, the most advanced are adeno-associated vectors that, while being safe and ensuring long-lasting transgene expression, have a very limited cargo capacity. In contrast, herpes simplex virus-based amplicon vectors can host huge amounts of foreign DNA, but concerns exist about their safety and ability to express transgenes long-term. We aimed at modulating and prolonging amplicon-induced transgene expression kinetics in vivo using different promoters and preventing transgene silencing. To pursue the latter, we deleted bacterial DNA sequences derived from vector construction and shielded the transgene cassette using AT-rich and insulator-like sequences (SAm technology). We employed luciferase and GFP as reporter genes. To determine transgene expression kinetics, we injected vectors in the hippocampus of mice that were longitudinally scanned for bioluminescence for 6 months. To evaluate safety, we analyzed multiple markers of damage and performed patch clamp electrophysiology experiments. All vectors proved safe, and we managed to modulate the duration of transgene expression, up to obtaining a stable, long-lasting expression using the SAm technology. Therefore, these amplicon vectors represent a flexible, efficient, and safe tool for gene delivery in the brain.