Dietary Protein Source Influences Brain Inflammation and Memory in a Male Senescence-Accelerated Mouse Model of Dementia.

Dementia is a pathological condition characterized by a decline in memory, as well as in other cognitive and social functions. The cellular and molecular mechanisms of brain damage in dementia are not completely understood; however, neuroinflammation is involved. Evidence suggests that chronic inflammation may impair cognitive performance and that dietary protein source may differentially influence this process. Dietary protein source has previously been shown to modify systemic inflammation in mouse models. Thus, we aimed to investigate the effect of chronic dietary protein source substitution in an ageing and dementia male mouse model, the senescence-accelerated mouse-prone 8 (SAMP8) model. We observed that dietary protein source differentially modified memory as shown by inhibitory avoidance testing at 4 months of age. Also, dietary protein source differentially modified neuroinflammation and gliosis in male SAMP8 mice. Our results suggest that chronic dietary protein source substitution may influence brain ageing and memory-related mechanisms in male SAMP8 mice. Moreover, the choice of dietary protein source in mouse diets for experimental purposes may need to be carefully considered when interpreting results.

Sarm1 deletion suppresses TDP-43-linked motor neuron degeneration and cortical spine loss.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative condition that primarily affects the motor system and shares many features with frontotemporal dementia (FTD). Evidence suggests that ALS is a 'dying-back' disease, with peripheral denervation and axonal degeneration occurring before loss of motor neuron cell bodies. Distal to a nerve injury, a similar pattern of axonal degeneration can be seen, which is mediated by an active axon destruction mechanism called Wallerian degeneration. Sterile alpha and TIR motif-containing 1 (Sarm1) is a key gene in the Wallerian pathway and its deletion provides long-term protection against both Wallerian degeneration and Wallerian-like, non-injury induced axonopathy, a retrograde degenerative process that occurs in many neurodegenerative diseases where axonal transport is impaired. Here, we explored whether Sarm1 signalling could be a therapeutic target for ALS by deleting Sarm1 from a mouse model of ALS-FTD, a TDP-43Q331K, YFP-H double transgenic mouse. Sarm1 deletion attenuated motor axon degeneration and neuromuscular junction denervation. Motor neuron cell bodies were also significantly protected. Deletion of Sarm1 also attenuated loss of layer V pyramidal neuronal dendritic spines in the primary motor cortex. Structural MRI identified the entorhinal cortex as the most significantly atrophic region, and histological studies confirmed a greater loss of neurons in the entorhinal cortex than in the motor cortex, suggesting a prominent FTD-like pattern of neurodegeneration in this transgenic mouse model. Despite the reduction in neuronal degeneration, Sarm1 deletion did not attenuate age-related behavioural deficits caused by TDP-43Q331K. However, Sarm1 deletion was associated with a significant increase in the viability of male TDP-43Q331K mice, suggesting a detrimental role of Wallerian-like pathways in the earliest stages of TDP-43Q331K-mediated neurodegeneration. Collectively, these results indicate that anti-SARM1 strategies have therapeutic potential in ALS-FTD.

Microglial overexpression of fALS-linked mutant SOD1 induces SOD1 processing impairment, activation and neurotoxicity and is counteracted by the autophagy inducer trehalose.

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease. Mutations in the gene encoding copper/zinc superoxide dismutase-1 (SOD1) are responsible for most familiar cases, but the role of mutant SOD1 protein dysfunction in non-cell autonomous neurodegeneration, especially in relation to microglial activation, is still unclear. Here, we focused our study on microglial cells, which release SOD1 also through exosomes. We observed that in rat primary microglia the overexpression of the most-common SOD1 mutations linked to fALS (G93A and A4V) leads to SOD1 intracellular accumulation, which correlates to autophagy dysfunction and microglial activation. In primary contact co-cultures, fALS mutant SOD1 overexpression by microglial cells appears to be neurotoxic by itself. Treatment with the autophagy-inducer trehalose reduced mutant SOD1 accumulation in microglial cells, decreased microglial activation and abrogated neurotoxicity in the co-culture model. These data suggest that i) the alteration of the autophagic pathway due to mutant SOD1 overexpression is involved in microglial activation and neurotoxicity; ii) the induction of autophagy with trehalose reduces microglial SOD1 accumulation through proteasome degradation and activation, leading to neuroprotection. Our results provide a novel contribution towards better understanding key cellular mechanisms in non-cell autonomous ALS neurodegeneration.

Nutritional and Pharmacological Strategies to Regulate Microglial Polarization in Cognitive Aging and Alzheimer's Disease.

The study of microglia, the immune cells of the brain, has experienced a renaissance after the discovery of microglia polarization. In fact, the concept that activated microglia can shift into the M1 pro-inflammatory or M2 neuroprotective phenotypes, depending on brain microenvironment, has completely changed the understanding of microglia in brain aging and neurodegenerative diseases. Microglia polarization is particularly important in aging since an increased inflammatory status of body compartments, including the brain, has been reported in elderly people. In addition, inflammatory markers, mainly derived from activated microglia, are widely present in neurodegenerative diseases. Microglial inflammatory dysfunction, also linked to microglial senescence, has been extensively demonstrated and associated with cognitive impairment in neuropathological conditions related to aging. In fact, microglia polarization is known to influence cognitive function and has therefore become a main player in neurodegenerative diseases leading to dementia. As the life span of human beings increases, so does the prevalence of cognitive dysfunction. Thus, therapeutic strategies aimed to modify microglia polarization are currently being developed. Pharmacological approaches able to shift microglia from M1 pro-inflammatory to M2 neuroprotective phenotype are actually being studied, by acting on many different molecular targets, such as glycogen synthase kinase-3 (GSK3) ß, AMP-activated protein kinase (AMPK), histone deacetylases (HDACs), etc. Furthermore, nutritional approaches can also modify microglia polarization and, consequently, impact cognitive function. Several bioactive compounds normally present in foods, such as polyphenols, can have anti-inflammatory effects on microglia. Both pharmacological and nutritional approaches seem to be promising, but still need further development. Here we review recent data on these approaches and propose that their combination could have a synergistic effect to counteract cognitive aging impairment and Alzheimer's disease (AD) through immunomodulation of microglia polarization, i.e., by driving the shift of activated microglia from the pro-inflammatory M1 to the neuroprotective M2 phenotype.

Zinc supplementation in rats impairs hippocampal-dependent memory consolidation and dampens post-traumatic recollection of stressful event.

Zinc is a trace element important for synaptic plasticity, learning and memory. Zinc deficiency, both during pregnancy and after birth, impairs cognitive performance and, in addition to memory deficits, also results in alterations of attention, activity, neuropsychological behavior and motor development. The effects of zinc supplementation on cognition, particularly in the adult, are less clear. We demonstrate here in adult rats, that 4 week-long zinc supplementation given by drinking water, and approximately doubling normal daily intake, strongly impairs consolidation of hippocampal-dependent memory, tested through contextual fear conditioning and inhibitory avoidance. Furthermore, the same treatment started after memory consolidation of training for the same behavioral tests, substantially dampens the recall of the stressful event occurred 4 weeks before. A molecular correlate of the amnesic effect of zinc supplementation is represented by a dysregulated function of GSK-3ß in the hippocampus, a kinase that participates in memory processes. The possible relevance of these data for humans, in particular regarding post-traumatic stress disorders, is discussed in view of future investigation.

Changing paradigm to target microglia in neurodegenerative diseases: from anti-inflammatory strategy to active immunomodulation.

INTRODUCTION: The importance of microglia in most neurodegenerative pathologies, from Parkinson's disease to amyotrophic lateral sclerosis and Alzheimer's disease, is increasingly recognized. Until few years ago, microglial activation in pathological conditions was considered dangerous to neurons due to its causing inflammation. Today we know that these glial cells also play a crucial physiological and neuroprotective role, which is altered in neurodegenerative conditions. AREAS COVERED: The neuroinflammatory hypothesis for neurodegenerative diseases has led to the trial of anti-inflammatory agents as therapeutics with largely disappointing results. New information about the physiopathological role of microglia has highlighted the importance of immunomodulation as a potential new therapeutic approach. This review summarizes knowledge on microglia as a potential therapeutic target in the most common neurodegenerative diseases, with focus on compounds directed toward the modulation of microglial immune response through specific molecular pathways. EXPERT OPINION: Here we support the innovative concept of targeting microglial cells by modulating their activity, rather than simply trying to counteract their inflammatory neurotoxicity, as a potential therapeutic approach for neurodegenerative diseases. The advantage of this therapeutic approach could be to reduce neuroinflammation and toxicity, while at the same time strengthening intrinsic neuroprotective properties of microglia and promoting neuroregeneration.

Neuronal Regulation of Neuroprotective Microglial Apolipoprotein E Secretion in Rat In Vitro Models of Brain Pathophysiology.

Apolipoprotein E (ApoE) is mainly secreted by glial cells and is involved in many brain functions, including neuronal plasticity, ß-amyloid clearance, and neuroprotection. Microglia--the main immune cells of the brain--are one source of ApoE, but little is known about the physiologic regulation of microglial ApoE secretion by neurons and whether this release changes under inflammatory or neurodegenerative conditions. Using rat primary neural cell cultures, we show that microglia release ApoE through a Golgi-mediated secretion pathway and that ApoE progressively accumulates in neuroprotective microglia-conditioned medium. This constitutive ApoE release is negatively affected by microglial activation both with lipopolysaccharide and with ATP. Microglial ApoE release is stimulated by neuron-conditioned media and under coculture conditions. Neuron-stimulated microglial ApoE release is mediated by serine and glutamate through N-methyl-D-aspartate receptors and is differently regulated by activation states (i.e. lipopolysaccharide vs ATP) and by 6-hydroxydopamine. Microglial ApoE silencing abrogated protection of cerebellar granule neurons against 6-hydroxydopamine toxicity in cocultures, indicating that microglial ApoE release is neuroprotective. Our findings shed light on the reciprocal cross-talk between neurons and microglia that is crucial for normal brain functions. They also open the way for the identification of possible pharmacologic targets that can modulate neuroprotective microglial ApoE release under pathologic conditions.

The transcription factor CCAAT enhancer-binding protein ß protects rat cerebellar granule neurons from apoptosis through its transcription-activating isoforms.

CCAAT enhancer-binding protein ß is a transcription factor that is involved in many brain processes, although its role in neuronal survival/death remains unclear. By using primary cultures of rat cerebellar granule neurons, we have shown here that CCAAT enhancer-binding protein ß is present as all of its isoforms: the transcriptional activators liver activator proteins 1 and 2, and the transcriptional inhibitor liver inhibitory protein. We have also shown that liver activator protein 1 undergoes post-translational modifications, such as phosphorylation and sumoylation. These isoforms have different subcellular localizations, liver activator protein 2 being found in the cytosolic fraction only, liver inhibitory protein in the nucleus only, and liver activator protein 1 in both fractions. Through neuronal apoptosis induction by shifting mature cerebellar granule neurons to low-potassium medium, we have demonstrated that nuclear liver activator protein 1 expression decreases and its phosphorylation disappears, whereas liver inhibitory protein levels increase in the nuclear fraction, suggesting a pro-survival role for liver activator protein transcriptional activation and a pro-apoptotic role for liver inhibitory protein transcriptional inhibition. To confirm this, we transfected cerebellar granule neurons with plasmids expressing liver activator protein 1, liver activator protein 2, or liver inhibitory protein respectively, and observed that both liver activator proteins, which increase CCAAT-dependent transcription, but not liver inhibitory protein, counteracted apoptosis, thus demonstrating the pro-survival role of liver activator proteins. These data significantly improve our current understanding of the role of CCAAT enhancer-binding protein ß in neuronal survival/apoptosis.

Copper-zinc superoxide dismutase (SOD1) is released by microglial cells and confers neuroprotection against 6-OHDA neurotoxicity.

Microglial-neuronal interactions are essential for brain physiopathology. In this framework, recent data have changed the concept of microglia from essentially macrophagic cells to crucial elements in maintaining neuronal homeostasis and function through the release of neuroprotective molecules. Using proteomic analysis, here we identify copper-zinc superoxide dismutase (SOD1) as a protein produced and released by cultured rat primary microglia. Evidence for a neuroprotective role of microglia-derived SOD1 resulted from experiments in which primary cerebellar granule neurons (CGNs) were exposed to the dopaminergic toxin 6-hydroxydopamine (6-OHDA). Microglial conditioned medium, in which SOD1 had accumulated, protected CGNs from degeneration, and neuroprotection was abrogated by SOD1 inhibitors. These effects were replicated when exogenous SOD1 was added to a nonconditioned medium. SOD1 neuroprotective action was mediated by increased cell calcium from an external source. Further experiments demonstrated the specificity of SOD1 neuroprotection against 6-OHDA compared to other types of neurotoxic challenges. SOD1, constitutively produced and released by microglia through a lysosomal secretory pathway, is identified here for the first time as an essential component of neuroprotection mediated by microglia. This novel information is relevant to stimulating further studies of microglia-mediated neuroprotection in in vivo models of neurodegenerative diseases.

Nitric oxide control of proliferation in nerve cells and in tumor cells of nervous origin.

Recent evidence suggests that nitric oxide (NO) has a remarkable anti-proliferative action towards dividing neural precursor cells as well as towards cells giving rise to neural-derived tumors. The present paper summarizes essential literature-derived information on this issue and provides novel experimental evidence for these NO-mediated actions regarding a well characterized population of neuronal precursors, the cerebellar granule cell precursors and a cell line of medulloblastoma, a pediatric tumor originating from these same precursor cells undergoing deregulated proliferation. Evidence is presented regarding the NO-mediated regulation of proliferation of neuronal precursor cells both during developmental and adult neurogenesis. Then, the role of NO in the control of proliferation of neural-derived tumor cells, such as PC12 and neuroblastoma cells, is discussed. Novel experimental data are provided documenting the anti-proliferative action of NO towards basal and mitogen-stimulated division of rat cerebellar granule cell precursors, as well as towards medulloblastoma DAOY cells. Finally, some molecular correlates of NO action on cell cycle regulation are discussed. Overall, the data presented and discussed here highlight similarities at the molecular level between physiologic processes regulating normal proliferation of neural precursors and pathologic deregulation of these processes leading to tumor formation.

Transcriptional profiling in the lumbar spinal cord of a mouse model of amyotrophic lateral sclerosis: a role for wild-type superoxide dismutase 1 in sporadic disease?

Mice bearing mutations of copper-zinc-superoxide dismutase recapitulate spinal cord motor neuron degeneration and disease progression occurring in human amyotrophic lateral sclerosis. We have investigated the relationship between disease progression and altered gene expression by comparing the transcriptional profiles in lumbar spinal cord, fronto-parietal cortex and hippocampus of mutant G93A-SOD1, wild-type SOD1 transgenic and non-transgenic mice. Gene expression was evaluated at 55 and 110 days of age, representing pre-symptomatic and advanced disease stages of G93A mice, respectively. Whereas no significant variations were detectable in cortical and hippocampal areas, several mutation-related changes were detected in the lumbar spinal cord at the symptomatic stage, consistent with a condition of neuronal distress. Also, at both ages, we found a number of transgene-related changes, i.e. variations occurring in both transgenic groups independently of the G93A mutation, with wild-type SOD1- and G93A-SOD1-overexpressing mice displaying global transcriptional similarity at 110 days of age. Some of the changes in common between the two transgenic groups involve genes implicated in oxidative stress, inflammation, spinocerebellar degeneration and other neurodegenerative disorders. The finding that gene expressional alterations potentially associated to cellular distress are shared by wild-type and mutant human SOD1-overexpressing mice raises the possibility that mutated (in familial ALS) or otherwise dysregulated (in sporadic ALS) SOD1 expression is a common pathogenetic substrate of the disease.

Long-term dietary administration of valproic acid does not affect, while retinoic acid decreases, the lifespan of G93A mice, a model for amyotrophic lateral sclerosis.

Mice bearing the mutated gene for Cu/Zn superoxide dismutase (G93A) are a good model for human amyotrophic lateral sclerosis (ALS). They develop progressive limb paralysis paralleled by loss of motor neurons of the cervical and lumbar spinal cord, which starts at 3-3.5 months of age and ends with death at 4-5 months. Several treatments have been attempted to delay clinical symptoms and to extend lifespan, and some have had modest beneficial effects. One such treatment, based on long-term administration of valproic acid (VPA), resulted in controversial results. We report here that, while dietary supplementation with high VPA dosage slows down motor neuron death, as assessed by measurement of a specific marker for cholinergic neurons in the spinal cord, it has no significant effect on lifespan. Recently, the hypothesis has been put forward that a deficiency of retinoic acid (RA) and its signaling may have a role in ALS. We report that long-term dietary supplementation with RA has no effect on the decrease of the cholinergic marker in the spinal cord, but it significantly shortens lifespan of G93A mice.

Regional and temporal alterations of ODC/polyamine system during ALS-like neurodegenerative motor syndrome in G93A transgenic mice.

Natural polyamines (putrescine, spermidine and spermine) are ubiquitous molecules known to regulate a number of physiological processes and suspected to play a role also in various pathological conditions. Changes in polyamine levels and in their biosynthetic enzymes have been described for some neurodegenerative diseases but the available data are incomplete and somewhat contradictory. We report here alterations of the key enzyme of the polyamine pathway, ornithine decarboxylase (ODC) catalytic activity and polyamine levels in different CNS areas from SOD1 G39A transgenic mice, an animal model for amyotrophic lateral sclerosis (ALS). ODC catalytic activity, was found significantly increased both in the cervical and lumbar spinal cord and, to a lesser extent in the brain stem of transgenic mice at a symptomatic stage of the disease (125-day-old mice), while no differences were present at a pre-symptomatic stage (55-day-old mice). In parallel with the increase of ODC activity putrescine levels were several times increased in both cervical and lumbar spinal cord and in the brain stem of 125-day-old SOD1 G39A mice. Higher order polyamines were not increased except for a significant increase of spermidine in the cervical spinal cord. The present data demonstrate considerable alterations of the ODC/polyamine system in a reliable animal model of ASL, consistent with their role in neurodegeneration and in particular in motor neuron diseases.

Neurochemical correlates of differential neuroprotection by long-term dietary creatine supplementation.

Dietary supplementation with creatine has proven to be beneficial in models of acute and chronic neurodegeneration. We report here data on the neurochemical correlates of differential protection of long-term creatine supplementation in two models of excitotoxicity in rats, as well as in the mouse model for ALS (G93A mice). In rats, the fall in cholinergic and GABAergic markers due to the excitotoxic death of intrinsic neurons caused by intrastriatal infusion of the neurotoxin, ibotenic acid, was significantly prevented by long-term dietary supplementation with creatine. On the contrary, creatine was unable to recover a cholinergic marker in the cortex of rats subjected to the excitotoxic death of the cholinergic basal forebrain neurons. In G93A mice, long-term creatine supplementation marginally but significantly increased mean lifespan, as previously observed by others, and reverted the cholinergic deficit present in some forebrain areas at an intermediate stage of the disease. In both rats and mice, creatine supplementation increased the activity of the GABAergic enzyme, glutamate decarboxylase, in the striatum but not in other brain regions. The present data point at alterations of neurochemical parameters marking specific neuronal populations, as a useful way to evaluate neuroprotective effects of long-term creatine supplementation in animal models of neurodegeneration.

Disease-related regressive alterations of forebrain cholinergic system in SOD1 mutant transgenic mice.

Transgenic mice carrying the human mutated SOD1 gene with a glycine/alanine substitution at codon 93 (G93A) are a widely used model for the fatal human disease amyotrophic lateral sclerosis (ALS). In these transgenic mice, we carried out a neurochemical study not only restricted to the primarily affected regions, the cervical and lumbar segments of the spinal cord, but also to several other brain regions. At symptomatic (110 and 125 days of age), but not at pre-symptomatic (55 days of age) stages, we found significant decreases in catalytic activity of the cholinergic enzyme, choline acetyltransferase (ChAT) in the hippocampus, olfactory cortex and fronto-parietal cortex. In parallel, we observed a decreased number of basal forebrain cholinergic neurons projecting to these areas. No alterations of the cholinergic markers were noticed in the striatum and the cerebellum. A widespread marker for GABAergic neurons, glutamate decarboxylase (GAD), was unaffected in all the areas examined. Alteration of cholinergic markers in forebrain areas was paralleled by concomitant alterations in the spinal cord and brainstem, as a consequence of progressive apoptotic elimination of cholinergic motor neuron. Gestational supplementation of choline, while able to result in long-term enhancement of cholinergic activity, did not improve transgenic mice lifespan nor counteracted cholinergic impairment in brain regions and spinal cord.