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Many lines of evidence have highlighted the role played by heterogeneous nuclear ribonucleoproteins in amyotrophic lateral sclerosis. In this study, we have aimed to identify transcripts co-regulated by TAR DNA-binding protein 43 kDa and highly conserved heterogeneous nuclear ribonucleoproteins which have been previously shown to regulate TAR DNA-binding protein 43 kDa toxicity (deleted in azoospermia-associated protein 1, heterogeneous nuclear ribonucleoprotein -Q, -D, -K and -U). Using the transcriptome analyses, we have uncovered that Nitric Oxide Synthase 1 Adaptor Protein mRNA is a direct TAR DNA-binding protein 43 kDa target, and in flies, its modulation alone can rescue TAR DNA-binding protein 43 kDa pathology. In primary mouse cortical neurons, we show that TAR DNA-binding protein 43 kDa mediated downregulation of Nitric Oxide Synthase 1 Adaptor Protein expression strongly affects the NMDA-receptor signalling pathway. In human patients, the downregulation of Nitric Oxide Synthase 1 Adaptor Protein mRNA strongly correlates with TAR DNA-binding protein 43 kDa proteinopathy as measured by cryptic Stathmin-2 and Unc-13 homolog A cryptic exon inclusion. Overall, our results demonstrate that Nitric Oxide Synthase 1 Adaptor Protein may represent a novel disease-relevant gene, potentially suitable for the development of new therapeutic strategies.

Amyotrophic Lateral Sclerosis (ALS) is considered the prototype of motor neuron disease, characterized by motor neuron loss and muscle waste. A well-established pathogenic hallmark of ALS is mitochondrial failure, leading to bioenergetic deficits. So far, pharmacological interventions for the disease have proven ineffective. Trimetazidine (TMZ) is described as a metabolic modulator acting on different cellular pathways. Its efficacy in enhancing muscular and cardiovascular performance has been widely described, although its molecular target remains elusive. We addressed the molecular mechanisms underlying TMZ action on neuronal experimental paradigms. To this aim, we treated murine SOD1G93A-model-derived primary cultures of cortical and spinal enriched motor neurons, as well as a murine motor-neuron-like cell line overexpressing SOD1G93A, with TMZ. We first characterized the bioenergetic profile of the cell cultures, demonstrating significant mitochondrial dysfunction that is reversed by acute TMZ treatments. We then investigated the effect of TMZ in promoting autophagy processes and its impact on mitochondrial morphology. Finally, we demonstrated the effectiveness of TMZ in terms of the mitochondrial functionality of ALS-rpatient-derived peripheral blood mononuclear cells (PBMCs). In summary, our results emphasize the concept that targeting mitochondrial dysfunction may represent an effective therapeutic strategy for ALS. The findings demonstrate that TMZ enhances mitochondrial performance in motor neuron cells by activating autophagy processes, particularly mitophagy. Although further investigations are needed to elucidate the precise molecular pathways involved, these results hold critical implications for the development of more effective and specific derivatives of TMZ for ALS treatment. [ABSTRACT FROM AUTHOR]

The modulation of synaptic strength associated with learning is post-synaptically regulated by changes in density and shape of dendritic spines. The transcription factor CREB (cAMP response element binding protein) is required for memory formation and in vitro dendritic spine rearrangements, but its role in learning-induced remodeling of neurons remains elusive. Using transgenic mice conditionally expressing a dominant-negative CREB (CREBS133A: mCREB) mutant, we found that inhibiting CREB function does not alter spine density, spine morphology, and levels of polymerized actin in naive CA1 neurons. CREB inhibition, however, impaired contextual fear conditioning and produced a learning-induced collapse of spines associated with a blockade of learning-dependent increase in actin polymerization. Blocking mCREB expression with doxycycline rescued memory and restored a normal pattern of learning-induced spines, demonstrating that CREB controls structural adaptations of neurons selectively involved in memory formation.

Degeneration of cortical and spinal motor neurons is the typical feature of amyotrophic lateral sclerosis (ALS), a progressive neurodegenerative disease for which a pathogenetic role for the Cu/Zn superoxide dismutase (SOD1) has been demonstrated. Mice overexpressing a mutated form of the SOD1 gene (SOD1G93A) develop a syndrome that closely resembles the human disease. The SOD1 mutations confer to this enzyme a “gain-of-function,” leading to increased production of reactive oxygen species. Several oxidants induce tyrosine phosphorylation through direct stimulation of kinases and/or phosphatases. In this study, we analyzed the activities of src and fyn tyrosine kinases and of protein tyrosine phosphatases in synaptosomal fractions prepared from the motor cortex and spinal cord of transgenic mice expressing SOD1G93A. We found that (i) protein phosphotyrosine level is increased, (ii) src and fyn activities are upregulated, and (iii) the activity of tyrosine phosphatases, including the striatal-enriched tyrosine phosphatase (STEP), is significantly decreased. Moreover, the NMDA receptor (NMDAR) subunit GluN2B tyrosine phosphorylation was upregulated in SOD1G93A. Tyrosine phosphorylation of GluN2B subunits regulates the NMDAR function and the recruitment of downstream signaling molecules. Indeed, we found that proline-rich tyrosine kinase 2 (Pyk2) and ERK1/2 kinase are upregulated in SOD1G93A mice. These results point out an involvement of tyrosine kinases and phosphatases in the pathogenesis of ALS. [ABSTRACT FROM AUTHOR]

Abstract: Although amyotrophic lateral sclerosis (ALS) is mainly considered as a motor disease, extramotor neural and cognitive alterations have also been reported in ALS patients. There is evidence that mutations in the Cu/Zn superoxide dismutase (SOD1) gene are implicated in about 20% of familiar ALS and transgenic mice overexpressing the human Cu/Zn superoxide dismutase (GLY93 → ALA) mutation show an ALS-like phenotype. However, while motor behavior has been extensively analyzed in these mutants, little is known on their cognitive abilities. To characterize the pre-symptomatic cognitive profile of G93A+/+ mice, we estimated their capability to detect spatial novelty and examined several indexes of their hippocampal function. We found an enhancement of spatial abilities in mutant mice associated with (1) a higher expression of hippocampal AMPA subunit GluR1 mRNA and of GluR1 protein levels, and (2) an increased induction and maintenance of long-term potentiation (LTP) at Schaffer collateral-CA1 synapses. Thus, before leading to extensive neuronal excitotoxicity, the high endogenous levels of glutamate present in the brain of pre-symptomatic G93A+/+ mice could mediate site-specific molecular and synaptic changes providing favorable conditions to spatial information processing. These findings suggest that identification of pre-symptomatic behavioral changes in murine models of ALS may point to early neural abnormalities selectively associated with mutations in the Cu/Zn superoxide dismutase (SOD1) gene. [Copyright &y& Elsevier]

Abstract: Zn2+ is co-released at glutamatergic synapses throughout the central nervous system and acts as a neuromodulator for glutamatergic neurotransmission, as a key modulator of NMDA receptor functioning. Zn2+ is also implicated in the neurotoxicity associated with several models of acute brain injury and neurodegeneration. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting motor neurons in the spinal cord and cortex. In this study, we have investigated the modulatory role exerted by Zn2+ in NMDA-mediated neurotoxicity in either near-pure or mixed cortical cultured neurons obtained from either mice over-expressing the G93A mutant form of Cu/Zn superoxide dismutase (SOD1) human gene, a gene linked to familial ALS, or wild type (WT) mice. To that aim, SOD1G93A or WT cultures were exposed to either NMDA by itself or to Zn2+ prior to a toxic challenge with NMDA, and neuronal loss evaluated 24 h later. While we failed to observe any significant difference between NMDA and Zn2+/NMDA-mediated toxicity in mixed SOD1G93A or WT cortical cultures, different vulnerability to these toxic paradigms was found in near-pure neuronal cultures. In the WT near-pure neuronal cultures, a brief exposure to sublethal concentrations of Zn2+-enhanced NMDA receptor-mediated cell death, an effect that was far more pronounced in the SOD1G93A cultures. This increased excitotoxicity in SOD1G93A near-pure neuronal cultures appears to be mediated by a significant increase in NMDA-dependent rises of intraneuronal Ca2+ levels as well as enhanced production of cytosolic reactive oxygen species, while the injurious process seems to be unrelated to activation of nNOS or ERK1/2 pathways. This article is part of a Special Issue entitled ‘Trends in Neuropharmacology: In Memory of Erminio Costa’. [Copyright &y& Elsevier]

Although amyotrophic lateral sclerosis (ALS) is mainly considered as a motor disease, extramotor neural and cognitive alterations have also been reported in ALS patients. There is evidence that mutations in the Cu/Zn superoxide dismutase (SOD1) gene are implicated in about 20% of familiar ALS and transgenic mice overexpressing the human Cu/Zn superoxide dismutase (GLY(93) --> ALA) mutation show an ALS-like phenotype. However, while motor behavior has been extensively analyzed in these mutants, little is known on their cognitive abilities. To characterize the pre-symptomatic cognitive profile of G93A+/+ mice, we estimated their capability to detect spatial novelty and examined several indexes of their hippocampal function. We found an enhancement of spatial abilities in mutant mice associated with (1) a higher expression of hippocampal AMPA subunit GluR1 mRNA and of GluR1 protein levels, and (2) an increased induction and maintenance of long-term potentiation (LTP) at Schaffer collateral-CA1 synapses. Thus, before leading to extensive neuronal excitotoxicity, the high endogenous levels of glutamate present in the brain of pre-symptomatic G93A+/+ mice could mediate site-specific molecular and synaptic changes providing favorable conditions to spatial information processing. These findings suggest that identification of pre-symptomatic behavioral changes in murine models of ALS may point to early neural abnormalities selectively associated with mutations in the Cu/Zn superoxide dismutase (SOD1) gene.