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Mechanisms of tissue damage in Huntington's disease involve excitotoxicity, mitochondrial damage, and inflammation, including microglia activation. Immunomodulatory and anti-protein aggregation properties of tetracyclines were demonstrated in several disease models. In the present study, the neuroprotective and anti-inflammatory effects of the tetracycline doxycycline were investigated in the mouse model of HD disease R6/2. Transgenic mice were daily treated with doxycycline 20 mg/kg, starting from 4 weeks of age. After sacrifice, histological and immunohistochemical studies were performed. We found that doxycycline-treated R6/2 mice survived longer and displayed less severe signs of neurological dysfunction than the saline-treated ones. Primary outcome measures such as striatal atrophy, neuronal intranuclear inclusions, and the negative modulation of microglial reaction revealed a neuroprotective effect of the compound. Doxycycline provided a significantly increase of activated CREB and BDNF in the striatal neurons, along with a down modulation of neuroinflammation, which, combined, might explain the beneficial effects observed in this model. Our findings show that doxycycline treatment could be considered as a valid therapeutic approach for HD.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease due to an expansion of a trinucleotide repeats in IT15 gene encoding for the protein huntingtin. Motor dysfunction, cognitive decline, and psychiatric disorder are typical clinical signs of HD. In HD, mutated huntingtin causes a major loss of brain derived neurotrophic factor (BDNF), causing striatal atrophy. Moreover, a key involvement of BDNF was observed in the synaptic plasticity that controls the acquisition and/or consolidation of certain forms of memory. We studied changes in hippocampal BDNF and in CREB in the R6/2 mouse model of HD. Moreover, we investigated if the beneficial effects of systemically administered recombinant BDNF observed in the striatum and cortex had an effect also on the hippocampus. Osmotic minipumps that chronically released recombinant BDNF or saline solution from 4 weeks of age until euthanasia were implanted into R6/2 and wild type mice. Our data show that BDNF is severely decreased in the hippocampus of R6/2 mice, while BDNF treatment restored its physiological levels. Moreover, the chronic administration of recombinant BDNF promoted the increment of phosphorylated CREB protein. Our study demonstrates the involvement of hippocampus in the pathology of R6/2 model of HD and correlates the beneficial effects of BDNF administration with increased hippocampal levels of BDNF and pCREB.

Inflammation significantly impacts the progression of Huntington's disease (HD) and the mutant HTT protein determines a pro-inflammatory activation of microglia. Mesenchymal stem/stromal cells (MSC) from the amniotic membrane (hAMSC), and their conditioned medium (CM-hAMSC), have been shown to possess protective effects in vitro and in vivo in animal models of immune-based disorders and of traumatic brain injury, which have been shown to be mediated by their immunomodulatory properties. In this study, in the R6/2 mouse model for HD we demonstrate that mice treated with CM-hAMSC display less severe signs of neurological dysfunction than saline-treated ones. CM-hAMSC treatment significantly delayed the development of the hind paw clasping response during tail suspension, reduced deficits in rotarod performance, and decreased locomotor activity in an open field test. The effects of CM-hAMSC on neurological function were reflected in a significant amelioration in brain pathology, including reduction in striatal atrophy and the formation of striatal neuronal intranuclear inclusions. In addition, while no significant increase was found in the expression of BDNF levels after CM-hAMSC treatment, a significant decrease of microglia activation and inducible nitric oxide synthase levels were observed. These results support the concept that CM-hAMSC could act by modulating inflammatory cells, and more specifically microglia., (© 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

During multiple sclerosis (MS), a close link has been demonstrated to occur between inflammation and neuro-axonal degeneration, leading to the hypothesis that immune mechanisms may promote neurodegeneration, leading to irreversible disease progression. Energy deficits and inflammation-driven mitochondrial dysfunction seem to be involved in this process. In this work we investigated, by the use of striatal electrophysiological field-potential recordings, if the inflammatory process associated with experimental autoimmune encephalomyelitis (EAE) is able to influence neuronal vulnerability to the blockade of mitochondrial complex IV, a crucial component for mitochondrial activity responsible of about 90% of total cellular oxygen consumption. We showed that during the acute relapsing phase of EAE, neuronal susceptibility to mitochondrial complex IV inhibition is markedly enhanced. This detrimental effect was counteracted by the pharmacological inhibition of microglia, of nitric oxide (NO) synthesis and its intracellular pathway (involving soluble guanylyl cyclase, sGC, and protein kinase G, PKG). The obtained results suggest that mitochondrial complex IV exerts an important role in maintaining neuronal energetic homeostasis during EAE. The pathological processes associated with experimental MS, and in particular the activation of microglia and of the NO pathway, lead to an increased neuronal vulnerability to mitochondrial complex IV inhibition, representing promising pharmacological targets., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Poly (ADP-ribose) polymerases (PARPs) are enzymes that catalyze ADP-ribose units transfer from NAD to their substrate proteins. It has been observed that PARP-1 is able to increase both post-ischemic and excitotoxic neuronal death. In fact, we have previously shown that, INO-1001, a PARP-1 inhibitor, displays a neuroprotective effect in the R6/2 model of Huntington's disease (HD). In this study, we investigated the effects of PARP-1-inhibition on modulation of phosphorylated c-AMP response element binding protein (pCREB) and CREB-binding protein (CBP) localization in the different striatal neuronal subsets. Moreover, we studied the neurodegeneration of those interneurons that are particularly vulnerable to HD such as parvalbuminergic and calretininergic, and of other subclasses of interneurons that are known to be resistant, such as cholinergic and somatostatinergic interneurons. Transgenic mice were treated with INO-1001 (10 mg/Kg daily) starting from 4 weeks of age. Double-label immunofluorescence was performed to value the distribution of CBP in ubiquitinated Neuronal intranuclear inclusions (NIIs) in the striatum. INO-1001-treated and saline-treated brain sections were incubated with: goat anti-choline acetyl transferase; goat anti-nitric oxide synthase; mouse anti-parvalbumin and mouse anti-calretinin. Morphometric evaluation and cell counts were performed. Our study showed that the PARP inhibitor has a positive effect in sparing parvalbumin and calretinin-containing interneurons of the striatum, where CREB was upregulated. Moreover, INO-1001 promoted CBP localization into the nuclei of the R6/2 mouse. The sum of our data corroborates the previous observations indicating PARP inhibition as a possible therapeutic tool to fight HD.

We report that changes of phosphodiesterase-10A (PDE10A) can map widespread functional imbalance of basal ganglia circuits in a mouse model of DYT1 dystonia overexpressing mutant torsinA. PDE10A is a key enzyme in the catabolism of second messenger cAMP and cGMP, whose synthesis is stimulated by D1 receptors and inhibited by D2 receptors preferentially expressed in striatoentopeducuncular/substantia nigra or striatopallidal pathways, respectively. PDE10A was studied in control mice (NT) and in mice carrying human wild-type torsinA (hWT) or mutant torsinA (hMT). Quantitative analysis of PDE10A expression was assessed in different brain areas by rabbit anti-PDE10A antibody immunohistochemistry and Western blotting. PDE10A-dependent cAMP hydrolyzing activity and PDE10A mRNA were also assessed. Striatopallidal neurons were identified by rabbit anti-enkephalin antibody.In NT mice, PDE10A is equally expressed in medium spiny striatal neurons and in their projections to entopeduncular nucleus/substantia nigra and to external globus pallidus. In hMT mice, PDE10A content selectively increases in enkephalin-positive striatal neuronal bodies; moreover, PDE10A expression and activity in hMT mice, compared with NT mice, significantly increase in globus pallidus but decrease in entopeduncular nucleus/substantia nigra. Similar changes of PDE10A occur in hWT mice, but such changes are not always significant. However, PDE10A mRNA expression appears comparable among NT, hWT, and hMT mice.In DYT1 transgenic mice, the inverse changes of PDE10A in striatoentopeduncular and striatopallidal projections might result over time in an imbalance between direct and indirect pathways for properly focusing movement. The decrease of PDE10A in the striatoentopeduncular/nigral projections might lead to increased intensity and duration of D1-stimulated cAMP/cGMP signaling; conversely, the increase of PDE10A in the striatopallidal projections might lead to increased intensity and duration of D2-inhibited cAMP/cGMP signaling. SIGNIFICANCE STATEMENT In DYT1 transgenic mouse model of dystonia, PDE10A, a key enzyme in cAMP and cGMP catabolism, is downregulated in striatal projections to entopeduncular nucleus/substantia nigra, preferentially expressing D1 receptors that stimulate cAMP/cGMP synthesis. Conversely, in DYT1 mice, PDE10A is upregulated in striatal projections to globus pallidus, preferentially expressing D2 receptors that inhibit cAMP/cGMP synthesis. The inverse changes to PDE10A in striatoentopeduncular/substantia nigra and striatopallidal pathways might tightly interact downstream to dopamine receptors, likely resulting over time to increased intensity and duration respectively of D1-stimulated and D2-inhibited cAMP/cGMP signals. Therefore, PDE10A changes in the DYT1 model of dystonia can upset the functional balance of basal ganglia circuits, affecting direct and indirect pathways simultaneously., (Copyright © 2017 the authors 0270-6474/17/372113-13$15.00/0.)

Background: Advanced Parkinson's disease (PD) is characterized by massive degeneration of nigral dopaminergic neurons, dramatic motor and cognitive alterations, and presence of nigral Lewy bodies, whose main constituent is α-synuclein (α-syn). However, the synaptic mechanisms underlying behavioral and motor effects induced by early selective overexpression of nigral α-syn are still a matter of debate., Methods: We performed behavioral, molecular, and immunohistochemical analyses in two transgenic models of PD, mice transgenic for truncated human α-synuclein 1-120 and rats injected with the adeno-associated viral vector carrying wild-type human α-synuclein. We also investigated striatal synaptic plasticity by electrophysiological recordings from spiny projection neurons and cholinergic interneurons., Results: We found that overexpression of truncated or wild-type human α-syn causes partial reduction of striatal dopamine levels and selectively blocks the induction of long-term potentiation in striatal cholinergic interneurons, producing early memory and motor alterations. These effects were dependent on α-syn modulation of the GluN2D-expressing N-methyl-D-aspartate receptors in cholinergic interneurons. Acute in vitro application of human α-syn oligomers mimicked the synaptic effects observed ex vivo in PD models., Conclusions: We suggest that striatal cholinergic dysfunction, induced by a direct interaction between α-syn and GluN2D-expressing N-methyl-D-aspartate receptors, represents a precocious biological marker of the disease., (Copyright © 2016 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

Cognitive impairment is common in multiple sclerosis (MS). Unfortunately, the synaptic and molecular mechanisms underlying MS-associated cognitive dysfunction are largely unknown. We explored the presence and the underlying mechanism of cognitive and synaptic hippocampal dysfunction during the remission phase of experimental MS. Experiments were performed in a chronic-relapsing experimental autoimmune encephalomyelitis (EAE) model of MS, after the resolution of motor deficits. Immunohistochemistry and patch-clamp recordings were performed in the CA1 hippocampal area. The hole-board was utilized as cognitive/behavioural test. In the remission phase of experimental MS, hippocampal microglial cells showed signs of activation, CA1 hippocampal synapses presented an impaired long-term potentiation (LTP) and an alteration of spatial tests became evident. The activation of hippocampal microglia mediated synaptic and cognitive/behavioural alterations during EAE. Specifically, LTP blockade was found to be caused by the reactive oxygen species (ROS)-producing enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. We suggest that in the remission phase of experimental MS microglia remains activated, causing synaptic dysfunctions mediated by NADPH oxidase. Inhibition of microglial activation and NADPH oxidase may represent a promising strategy to prevent neuroplasticity impairment associated with active neuro-inflammation, with the aim to improve cognition and counteract MS disease progression.

Poly (ADP-ribose) polymerase 1 (PARP-1) is a nuclear enzyme that is involved in physiological processes as DNA repair, genomic stability, and apoptosis. Moreover, published studies demonstrated that PARP-1 mediates necrotic cell death in response to excessive DNA damage under certain pathological conditions. In Huntington's disease brains, PARP immunoreactivity was described in neurons and in glial cells, thereby suggesting the involvement of apoptosis in HD. In this study, we sought to determine if the PARP-1 inhibitor exerts a neuroprotective effect in R6/2 mutant mice, which recapitulates, in many aspects, human HD. Transgenic mice were treated with the PARP-1 inhibitor INO-1001 mg/Kg daily starting from 4 weeks of age. After transcardial perfusion, histological and immunohistochemical studies were performed. We found that INO 1001-treated R6/2 mice survived longer and displayed less severe signs of neurological dysfunction than the vehicle treated ones. Primary outcome measures such as striatal atrophy, morphology of striatal neurons, neuronal intranuclear inclusions and microglial reaction confirmed a neuroprotective effect of the compound. INO-1001 was effective in significantly increasing activated CREB and BDNF in the striatal spiny neurons, which might account for the beneficial effects observed in this model. Our findings show that PARP-1 inhibition could be considered as a valid therapeutic approach for HD.

Mechanisms of gender-specific synaptic plasticity in the striatum, a brain region that controls motor, cognitive and psychiatric functions, remain unclear. Here we report that Rhes, a GTPase enriched in medium spiny neurons (MSNs) of striatum, alters the striatal cAMP/PKA signaling cascade in a gender-specific manner. While Rhes knockout (KO) male mice, compared to wild-type (WT) mice, had a significant basal increase of cAMP/PKA signaling pathway, the Rhes KO females exhibited a much stronger response of this pathway, selectively under the conditions of dopamine/adenosine-related drug challenge. Corticostriatal LTP defects are exclusively found in A2AR/D2R-expressing MSNs of KO females, compared to KO males, an effect that is abolished by PKA inhibitors but not by the removal of circulating estrogens. This suggests that the synaptic alterations found in KO females could be triggered by an aberrant A2AR/cAMP/PKA activity, but not due to estrogen-mediated effect. Consistent with increased cAMP signaling, D1R-mediated motor stimulation, haloperidol-induced catalepsy and caffeine-evoked hyper-activity are robustly enhanced in Rhes KO females compared to mutant males. Thus Rhes, a thyroid hormone-target gene, plays a relevant role in gender-specific synaptic and behavioral responses.

17β-estradiol (E2), a neurosteroid synthesized by P450-aromatase (ARO), modulates various brain functions. We characterized the role of the locally synthesized E2 on striatal long-term synaptic plasticity and explored possible interactions between E2 receptors (ERs) and dopamine (DA) receptors in the dorsal striatum of adult male rats. Inhibition of E2 synthesis or antagonism of ERs prevented the induction of long-term potentiation (LTP) in both medium spiny neurons (MSNs) and cholinergic interneurons (ChIs). Activation of a D1-like DA receptor/cAMP/PKA-dependent pathway restored LTP. In MSNs exogenous E2 reversed the effect of ARO inhibition. Also antagonism of M1 muscarinic receptors prevented the D1-like receptor-mediated restoration of LTP confirming a role for ChIs in controlling the E2-mediated LTP of MSNs. A novel striatal interaction, occurring between ERs and D1-like receptors in both MSNs and ChIs, might be critical to regulate basal ganglia physiology and to compensate synaptic alterations in Parkinson's disease.

Background: Bidirectional long-term plasticity at the corticostriatal synapse has been proposed as a central cellular mechanism governing dopamine-mediated behavioral adaptations in the basal ganglia system. Balanced activity of medium spiny neurons (MSNs) in the direct and the indirect pathways is essential for normal striatal function. This balance is disrupted in Parkinson's disease and in l-3,4-dihydroxyphenylalanine (l-DOPA)-induced dyskinesia (LID), a common motor complication of current pharmacotherapy of Parkinson's disease., Methods: Electrophysiological recordings were performed in mouse cortico-striatal slice preparation. Synaptic plasticity, such as long-term potentiation (LTP) and depotentiation, was investigated. Specific pharmacological inhibitors or genetic manipulations were used to modulate the Ras-extracellular signal-regulated kinase (Ras-ERK) pathway, a signal transduction cascade implicated in behavioral plasticity, and synaptic activity in different subpopulations of striatal neurons was measured., Results: We found that the Ras-ERK pathway, is not only essential for long-term potentiation induced with a high frequency stimulation protocol (HFS-LTP) in the dorsal striatum, but also for its reversal, synaptic depotentiation. Ablation of Ras-guanine nucleotide-releasing factor 1 (Ras-GRF1), a neuronal activator of Ras proteins, causes a specific loss of HFS-LTP in the medium spiny neurons in the direct pathway without affecting LTP in the indirect pathway. Analysis of LTP in animals with unilateral 6-hydroxydopamine lesions (6-OHDA) rendered dyskinetic with chronic L-DOPA treatment reveals a complex, Ras-GRF1 and pathway-independent, apparently stochastic involvement of ERK., Conclusions: These data not only demonstrate a central role for Ras-ERK signaling in striatal LTP, depotentiation, and LTP restored after L-DOPA treatment but also disclose multifaceted synaptic adaptations occurring in response to dopaminergic denervation and pulsatile administration of L-DOPA., (Copyright © 2015 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

Huntington's disease (HD) is an autosomal-dominant inherited neurodegenerative disorder characterized by motor dysfunction, cognitive decline, and emotional and psychiatric disturbances. The genetic mutation is characterized by a CAG expansion, resulting in the formation of a mutant huntingtin protein with an expanded polyglutamine repeat region. Mutated huntingtin has been shown to impair a number of physiological activities by interacting with several factors. In particular, cAMP response element-binding protein (CREB) and brain-derived neurotrophic factor (BDNF) are severely affected by mutant huntingtin. In this view, drugs targeted at counteracting CREB loss of function and BDNF decrease have been considered as powerful tools to treat HD. Recently, cyclic nucleotide phosphodiesterase (PDE) inhibitors have been used successfully to increase levels of CREB and BDNF in HD models. Indeed, PDE4, 5 or 10 inhibitors have been shown to afford neuroprotection and modulation of CREB and BDNF. In this review, we will summarize the data supporting the use of PDE inhibitors as the therapeutical approach to fight HD and we will discuss the possible mechanisms of action underlying these effects.