New insights on neurodegeneration triggered by iron accumulation: Intersections with neutral lipid metabolism, ferroptosis, and motor impairment.

Brain iron accumulation constitutes a pathognomonic indicator in several neurodegenerative disorders. Metal accumulation associated with dopaminergic neuronal death has been documented in Parkinson's disease. Through the use of in vivo and in vitro models, we demonstrated that lipid dysregulation manifests as a neuronal and glial response during iron overload. In this study, we show that cholesterol content and triacylglycerol (TAG) hydrolysis were strongly elevated in mice midbrain. Lipid cacostasis was concomitant with the loss of dopaminergic neurons, astrogliosis and elevated expression of α-synuclein. Exacerbated lipid peroxidation and markers of ferroptosis were evident in the midbrain from mice challenged with iron overload. An imbalance in the activity of lipolytic and acylation enzymes was identified, favoring neutral lipid hydrolysis, and consequently reducing TAG and cholesteryl ester levels. Notably, these observed alterations were accompanied by motor impairment in iron-treated mice. In addition, neuronal and glial cultures along with their secretomes were used to gain further insight into the mechanism underlying TAG hydrolysis and cholesterol accumulation as cellular responses to iron accumulation. We demonstrated that TAG hydrolysis in neurons is triggered by astrocyte secretomes. Moreover, we found that the ferroptosis inhibitor, ferrostatin-1, effectively prevents cholesterol accumulation both in neurons and astrocytes. Taken together, these results indicate that lipid disturbances occur in iron-overloaded mice as a consequence of iron-induced oxidative stress and depend on neuron-glia crosstalk. Our findings suggest that developing therapies aimed at restoring lipid homeostasis may lead to specific treatment for neurodegeneration associated with ferroptosis and brain iron accumulation.

Vitamin E Analog Trolox Attenuates MPTP-Induced Parkinson's Disease in Mice, Mitigating Oxidative Stress, Neuroinflammation, and Motor Impairment.

Trolox is a potent antioxidant and a water-soluble analog of vitamin E. It has been used in scientific studies to examine oxidative stress and its impact on biological systems. Trolox has been shown to have a neuroprotective effect against ischemia and IL-1ß-mediated neurodegeneration. In this study, we investigated the potential protective mechanisms of Trolox against a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson's disease mouse model. Western blotting, immunofluorescence staining, and ROS/LPO assays were performed to investigate the role of trolox against neuroinflammation, the oxidative stress mediated by MPTP in the Parkinson's disease (PD) mouse model (wild-type mice (C57BL/6N), eight weeks old, average body weight 25-30 g). Our study showed that MPTP increased the expression of α-synuclein, decreased tyrosine hydroxylase (TH) and dopamine transporter (DAT) levels in the striatum and substantia nigra pars compacta (SNpc), and impaired motor function. However, Trolox treatment significantly reversed these PD-like pathologies. Furthermore, Trolox treatment reduced oxidative stress by increasing the expression of nuclear factor erythroid-2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1). Lastly, Trolox treatment inhibited the activated astrocytes (GFAP) and microglia (Iba-1), also reducing phosphorylated nuclear factor-κB, (p-NF-κB) and tumor necrosis factor-alpha (TNF-α) in the PD mouse brain. Overall, our study demonstrated that Trolox may exert neuroprotection on dopaminergic neurons against MPTP-induced oxidative stress, neuroinflammation, motor dysfunction, and neurodegeneration.

Creatinine-to-cystatin C ratio estimates muscle mass correlating the markers of the patients with severe motor and intellectual disabilities.

AIM: The creatinine-to-cystatin C ratio (CCR) has been acknowledged as a potential marker of muscle mass. The purpose of the present study was to evaluate the relationship between CCR and nutritional status in a bioelectrical impedance analysis (BIA) of patients with severe motor and intellectual disabilities (SMID). METHODS: This study included 39 patients with SMID (17 males, 22 females) over 16 years of age were included retrospectively. CCR was calculated as serum creatinine (mg/dL)/cystatin C (mg/L) × 10. The BIA parameters such as the phase angle (PhA), fat free mass (FFM), appendicular skeletal muscle mass (ASM) and appendicular skeletal muscle mass index (ASMI) values were measured using BIA. Correlation analyses between CCR and the BIA parameters were conducted. RESULTS: The mean CCR is 4.47 ±â€¯1.34. Significant positive relationships between CCR and FFM, PhA, ASM, ASMI were identified (r = 0.3373, p = 0.0357. r = 0.4273, p = 0.0093. r = 0.5008, p = 0.0012. r = 0.4706, p = 0.0025 and r = 0.4751, p = 0.0022, respectively). CONCLUSIONS: The study indicated that CCR in the patients with SMID is a useful parameter that allows for the muscle mass to be estimated easily and accurately. This means that evaluating CCR could be used as a simple and important screening tool for PhA, FFM and muscle mass.

Intranasal delivery of exosomes from human adipose derived stem cells at forty-eight hours post injury reduces motor and cognitive impairments following traumatic brain injury.

The neuroprotective role of human adipose-derived stems cells (hASCs) has raised great interest in regenerative medicine due to their ability to modulate their surrounding environment. Our group has demonstrated that exosomes derived from hASC (hASCexo) are a cell-free regenerative approach to long term recovery following traumatic brain injury (TBI). Previously, we demonstrated the efficacy of exosome treatment with intravenous delivery at 3 h post TBI in rats. Here, we show efficacy of exosomes through intranasal delivery at 48 h post TBI in mice lengthening the therapeutic window of treatment and therefore increasing possible translation to clinical studies. Our findings demonstrate significant recovery of motor impairment assessed by an elevated body swing test in mice treated with exosomes containing MALAT1 compared to both TBI mice without exosomes and exosomes depleted of MALAT1. Significant cognitive improvement was seen in the reversal trial of 8 arm radial arm water maze in mice treated with exosomes containing MALAT1. Furthermore, cortical damage was significantly reduced in mice treated with exosomes containing MALAT1 as well as decreased MHCII+ staining of microglial cells. Mice without exosomes or treated with exosomes depleted of MALAT1 did not show similar recovery. Results demonstrate both inflammation related genes and NRTK3 (TrkC) are target genes modulated by hASC exosomes and further that MALAT1 in hASC exosomes regulates expression of full length TrkC thereby activating the MAPK pathway and promoting recovery. Exosomes are a promising therapeutic approach following TBI with a therapeutic window of at least 48 h and contain long noncoding RNA's, specifically MALAT1 that play a vital role in the mechanism of action.

Gain of toxic function by long-term AAV9-mediated SMN overexpression in the sensorimotor circuit.

The neurodegenerative disease spinal muscular atrophy (SMA) is caused by deficiency in the survival motor neuron (SMN) protein. Currently approved SMA treatments aim to restore SMN, but the potential for SMN expression beyond physiological levels is a unique feature of adeno-associated virus serotype 9 (AAV9)-SMN gene therapy. Here, we show that long-term AAV9-mediated SMN overexpression in mouse models induces dose-dependent, late-onset motor dysfunction associated with loss of proprioceptive synapses and neurodegeneration. Mechanistically, aggregation of overexpressed SMN in the cytoplasm of motor circuit neurons sequesters components of small nuclear ribonucleoproteins, leading to splicing dysregulation and widespread transcriptome abnormalities with prominent signatures of neuroinflammation and the innate immune response. Thus, long-term SMN overexpression interferes with RNA regulation and triggers SMA-like pathogenic events through toxic gain-of-function mechanisms. These unanticipated, SMN-dependent and neuron-specific liabilities warrant caution on the long-term safety of treating individuals with SMA with AAV9-SMN and the risks of uncontrolled protein expression by gene therapy.

Rifampicin ameliorates lipopolysaccharide-induced cognitive and motor impairments via inhibition of the TLR4/MyD88/NF-κB signaling pathway in mice.

OBJECTIVES: Aberrant microglial responses promote neuroinflammation in neurodegenerative diseases. However, rifampicin's effect on cognitive and motor sequelae of inflammation remains unknown. Therefore, we investigated whether rifampicin exerts neuroprotection against lipopolysaccharide (LPS)-induced cognitive and motor impairments. METHODS: A mouse model of LPS-induced cognitive and motor impairment was established. Adult C57BL/6 mice were injected intraperitoneally with 25 mg/kg rifampicin 30 min before intraperitoneal microinjection of LPS (750 µg/kg) daily until study end. Treatments and behavioral experiments were performed once daily for 7 days. Behavioral tests and pathological/biochemical assays were performed to evaluate LPS-induced damage to the hippocampus and substantia nigra (SN). RESULTS: Rifampicin attenuated LPS-induced cognitive and motor impairments, based on performance in the behavioral tests. Rifampicin suppressed the release of pro-inflammatory mediators, including tumor necrosis factor-α, interleukin-1ß, and prostaglandin E2 in the serum and nitric oxide (NO) in brain tissue, and cyclooxygenase-2 and inducible nitric oxide synthase levels. Immunofluorescence revealed that rifampicin inhibited LPS-induced microglial activation in the hippocampus and SN, thus protecting the neurons. Rifampicin inhibited the activation of the toll-like receptor 4 (TLR4)/myeloid differentiation primary response 88 (MyD88)/nuclear factor kappa B (NF-κB) signaling pathway. Rifampicin downregulated TLR4 and MyD88 protein levels and inhibited NF-κB inhibitor alpha and NF-κB inhibitor kinase beta phosphorylation, thus reducing p65 nuclear transfer by inhibiting NF-κB signaling activation in LPS-treated mice. CONCLUSION: Rifampicin protects against LPS-induced neuroinflammation and attenuates cognitive and motor impairments by inhibiting the TLR4/MyD88/NF-κB signaling pathway. Our findings might aid the development of novel therapies to treat progressive neurodegenerative diseases.

Polygala sabulosa A.W. Bennett extract mitigates motor and cognitive deficits in a mouse model of acute ischemia.

Stroke is considered one of the leading causes of death worldwide. The treatment is limited; however, the Brazilian flora has a great source of natural products with therapeutic potentials. Studies with the medicinal plant Polygala sabulosa W. Bennett provided evidence for its use as an anti-inflammatory and neuroprotective drug. In the case of ischemic stroke due to lack of oxygen, both acute and chronic inflammatory processes are activated. Thus, we hypothesized that P. sabulosa (HEPs) has the potential to treat the motor and cognitive deficits generated by ischemic stroke. Male mice were subjected to global ischemia for 60 min, followed by reperfusion and orally treated with HEPs (100 mg/kg in saline + 3% tween 20) twice a day (12 h apart) for 48 h starting 3 h after surgery. Motor skills were assessed using grip force and open field tasks. Hippocampi were then collected for mRNA quantification of the cytokines IL-1-ß and TNF-α levels. After 48 h of acute treatment, spatial reference memory was evaluated in a Morris water maze test for another group of animals. We show that HEPs treatment significantly prevented motor weakness induced by ischemia. Brain infarct area was reduced by 22.25% with downregulation of the levels of IL-1ß and TNF-α mRNA. Learning performance and memory ability on Morris water maze task were similar to the sham group. Our data demonstrates the neuroprotective properties of HEPs through its anti-inflammatory activities, which prevent motor and cognitive impairments, suggesting that HEPs may be an effective therapy for ischemic stroke.

Neuronal Ablation of CoA Synthase Causes Motor Deficits, Iron Dyshomeostasis, and Mitochondrial Dysfunctions in a CoPAN Mouse Model.

COASY protein-associated neurodegeneration (CoPAN) is a rare but devastating genetic autosomal recessive disorder of inborn error of CoA metabolism, which shares with pantothenate kinase-associated neurodegeneration (PKAN) similar features, such as dystonia, parkinsonian traits, cognitive impairment, axonal neuropathy, and brain iron accumulation. These two disorders are part of the big group of neurodegenerations with brain iron accumulation (NBIA) for which no effective treatment is available at the moment. To date, the lack of a mammalian model, fully recapitulating the human disorder, has prevented the elucidation of pathogenesis and the development of therapeutic approaches. To gain new insights into the mechanisms linking CoA metabolism, iron dyshomeostasis, and neurodegeneration, we generated and characterized the first CoPAN disease mammalian model. Since CoA is a crucial metabolite, constitutive ablation of the Coasy gene is incompatible with life. On the contrary, a conditional neuronal-specific Coasy knock-out mouse model consistently developed a severe early onset neurological phenotype characterized by sensorimotor defects and dystonia-like movements, leading to premature death. For the first time, we highlighted defective brain iron homeostasis, elevation of iron, calcium, and magnesium, together with mitochondrial dysfunction. Surprisingly, total brain CoA levels were unchanged, and no signs of neurodegeneration were present.

Impact of Aging on the 6-OHDA-Induced Rat Model of Parkinson's Disease.

Parkinson's disease (PD) is the second most common age-related neurodegenerative disorder. The neurodegeneration leading to incapacitating motor abnormalities mainly occurs in the nigrostriatal pathway due to the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). Several animal models have been developed not only to better understand the mechanisms underlying neurodegeneration but also to test the potential of emerging disease-modifying therapies. However, despite aging being the main risk factor for developing idiopathic PD, most of the studies do not use aged animals. Therefore, this study aimed at assessing the effect of aging in the unilateral 6-hydroxydopamine (6-OHDA)-induced animal model of PD. For this, female young adult and aged rats received a unilateral injection of 6-OHDA into the medial forebrain bundle. Subsequently, the impact of aging on 6-OHDA-induced effects on animal welfare, motor performance, and nigrostriatal integrity were assessed. The results showed that aging had a negative impact on animal welfare after surgery. Furthermore, 6-OHDA-induced impairments on skilled motor function were significantly higher in aged rats when compared with their younger counterparts. Nigrostriatal histological analysis further revealed an increased 6-OHDA-induced dopaminergic cell loss in the SNpc of aged animals when compared to young animals. Overall, our results demonstrate a higher susceptibility of aged animals to 6-OHDA toxic insult.

Combination therapy with astaxanthin and epidermal neural crest stem cells improves motor impairments and activates mitochondrial biogenesis in a rat model of spinal cord injury.

Spinal cord injury (SCI), a multifactorial disease, can lead to irreversible motor and sensory disabilities. Cell therapy in combination with pharmacological agents can be a promising approach to attenuate SCI damages. Epidermal neural crest stem cells (EPI-NCSCs) extracted from bulge hair follicle in adults are attractive candidates due to the possibility of autologous transplantation. This study evaluated the effect of EPI-NCSCs combined with astaxanthin (Ast), a potent antioxidant, on damages induced by SCI. Male rats were treated with Ast (0.2 mM) and EPI-NCSCs (106/10 µl PBS) alone and combined together after SCI contusion. Motor function was assessed by Basso, Beattie and Bresnahan (BBB) test on days 1, 3, 7, 14, 21, 28, 35 and 42 post-injury. Motor neurons number and myelin level were evaluated on days 14 and 42 using Nissl and Luxol Fast Blue staining. The gene expression of mitochondrial biogenesis involved factors (PGC1α, NRF1 and TFAM) was measured by qPCR. All treatments improved motor function, with the highest BBB score in Ast + Cell compared to Ast and Cell. Decreased motor neurons number and myelin level following SCI, were increased by Ast, Cell and Ast + Cell, but combination therapy significantly had a better effect. We observed reduction in PGC1α, NRF1, and TFAM expression in spinal tissue after SCI, and treatment with Cell and Ast + Cell significantly restored NRF1 and TFAM mRNA levels. These results suggested that Ast in combination with EPI-NCSCs has better effects on behavioral dysfunction, motor neuron loss and demyelination after SCI. These protective effects may be attributed to mitochondrial biogenesis activation.

Elavl3 is essential for the maintenance of Purkinje neuron axons.

Neuronal Elav-like (nElavl or neuronal Hu) proteins are RNA-binding proteins that regulate RNA stability and alternative splicing, which are associated with axonal and synaptic structures. nElavl proteins promote the differentiation and maturation of neurons via their regulation of RNA. The functions of nElavl in mature neurons are not fully understood, although Elavl3 is highly expressed in the adult brain. Furthermore, possible associations between nElavl genes and several neurodegenerative diseases have been reported. We investigated the relationship between nElavl functions and neuronal degeneration using Elavl3-/- mice. Elavl3-/- mice exhibited slowly progressive motor deficits leading to severe cerebellar ataxia, and axons of Elavl3-/- Purkinje cells were swollen (spheroid formation), followed by the disruption of synaptic formation of axonal terminals. Deficit in axonal transport and abnormalities in neuronal polarity was observed in Elavl3-/- Purkinje cells. These results suggest that nElavl proteins are crucial for the maintenance of axonal homeostasis in mature neurons. Moreover, Elavl3-/- mice are unique animal models that constantly develop slowly progressive axonal degeneration. Therefore, studies of Elavl3-/- mice will provide new insight regarding axonal degenerative processes.

SLC39A14 deficiency alters manganese homeostasis and excretion resulting in brain manganese accumulation and motor deficits in mice.

Solute carrier family 39, member 14 (SLC39A14) is a transmembrane transporter that can mediate the cellular uptake of zinc, iron, and manganese (Mn). Studies of Slc39a14 knockout (Slc39a14-/-) mice have documented that SLC39A14 is required for systemic growth, hepatic zinc uptake during inflammation, and iron loading of the liver in iron overload. The normal physiological roles of SLC39A14, however, remain incompletely characterized. Here, we report that Slc39a14-/- mice spontaneously display dramatic alterations in tissue Mn concentrations, suggesting that Mn is a main physiological substrate for SLC39A14. Specifically, Slc39a14-/- mice have abnormally low Mn levels in the liver coupled with markedly elevated Mn concentrations in blood and most other organs, especially the brain and bone. Radiotracer studies using 54Mn reveal that Slc39a14-/- mice have impaired Mn uptake by the liver and pancreas and reduced gastrointestinal Mn excretion. In the brain of Slc39a14-/- mice, Mn accumulated in the pons and basal ganglia, including the globus pallidus, a region susceptible to Mn-related neurotoxicity. Brain Mn accumulation in Slc39a14-/- mice was associated with locomotor impairments, as assessed by various behavioral tests. Although a low-Mn diet started at weaning was able to reverse brain Mn accumulation in Slc39a14-/- mice, it did not correct their motor deficits. We conclude that SLC39A14 is essential for efficient Mn uptake by the liver and pancreas, and its deficiency results in impaired Mn excretion and accumulation of the metal in other tissues. The inability of Mn depletion to correct the motor deficits in Slc39a14-/- mice suggests that the motor impairments represent lasting effects of early-life Mn exposure.

Glycine-Binding Site Stimulants of NMDA Receptors Alleviate Extrapyramidal Motor Disorders by Activating the Nigrostriatal Dopaminergic Pathway.

Dysfunction of the N-methyl-d-aspartate (NMDA) receptor has been implicated in the pathogenesis of schizophrenia. Although agonists for the glycine-binding sites of NMDA receptors have potential as new medication for schizophrenia, their modulation of antipsychotic-induced extrapyramidal side effects (EPS) has not yet been clarified. We herein evaluated the effects of glycine-binding site stimulants of NMDA receptors on antipsychotic-induced EPS in mice and rats. d-cycloserine (DCS) and d-serine significantly improved haloperidol (HAL)-induced bradykinesia in mice, whereas glycine showed no effects. Sodium benzoate, a d-amino acid oxidase inhibitor, also attenuated HAL-induced bradykinesia. Improvements in HAL-induced bradykinesia by DCS were antagonized by the NMDA antagonist dizocilpine or nitric oxide synthase inhibitor L-NG-Nitro-l-arginine methyl ester. In addition, DCS significantly reduced HAL-induced Fos expression in the dorsolateral striatum without affecting that in the nucleus accumbens. Furthermore, a microinjection of DCS into the substantia nigra pars compacta significantly inhibited HAL-induced EPS concomitant with elevations in dopamine release in the striatum. The present results demonstrated for the first time that stimulating the glycine-binding sites of NMDA receptors alleviates antipsychotic-induced EPS by activating the nigrostriatal dopaminergic pathway, suggesting that glycine-binding site stimulants are beneficial not only for efficacy, but also for side-effect management.

Interaction between subclinical doses of the Parkinson's disease associated gene, α-synuclein, and the pesticide, rotenone, precipitates motor dysfunction and nigrostriatal neurodegeneration in rats.

In most patients, Parkinson's disease is thought to emerge after a lifetime of exposure to, and interaction between, various genetic and environmental risk factors. One of the key genetic factors linked to this condition is α-synuclein, and the α-synuclein protein is pathologically associated with idiopathic cases. However, α-synuclein pathology is also present in presymptomatic, clinically "normal" individuals suggesting that environmental factors, such as Parkinson's disease-linked agricultural pesticides, may be required to precipitate Parkinson's disease in these individuals. In this context, the aim of this study was to assess the behavioural and neuropathological impact of exposing rats with a subclinical load of α-synuclein to subclinical doses of the organic pesticide, rotenone. Rats were randomly assigned to two groups for intra-nigral infusion of AAV2/5-GFP or AAV2/5-α-synuclein. Post viral motor function was assessed at 8, 10 and 12 weeks in the Corridor, Stepping and Whisker tests of lateralised motor function. At week 12, animals were performance-matched to receive a subsequent intra-striatal challenge of the organic pesticide rotenone (or its vehicle) to yield four final groups (Control, Rotenone, AAV2/5-α-synuclein and Combined). Behavioural testing resumed one week after rotenone surgery and continued for 5 weeks. We found that, when administered alone, neither intra-nigral AAV-α-synuclein nor intra-striatal rotenone caused sufficient nigrostriatal neurodegeneration to induce a significant motor impairment in their own right. However, when these were administered sequentially to the same rats, the interaction between the two Parkinsonian challenges significantly exacerbated nigrostriatal neurodegeneration which precipitated a pronounced impairment in motor function. These results indicate that exposing rats with a subclinical α-synuclein-induced pathology to the pesticide, rotenone, profoundly exacerbates their Parkinsonian neuropathology and dysfunction, and highlights the potential importance of this interaction in the etiology of, and in driving the pathogenesis of Parkinson's disease.

Decreased mitochondrial bioenergetics and calcium buffering capacity in the basal ganglia correlates with motor deficits in a nonhuman primate model of aging.

Altered mitochondrial function in the basal ganglia has been hypothesized to underlie cellular senescence and promote age-related motor decline. We tested this hypothesis in a nonhuman primate model of human aging. Six young (6-8 years old) and 6 aged (20-25 years old) female Rhesus monkeys (Macaca mulatta) were behaviorally characterized from standardized video records. Additionally, we measured mitochondrial bioenergetics along with calcium buffering capacity in the substantia nigra and putamen (PUT) from both age groups. Our results demonstrate that the aged animals had significantly reduced locomotor activity and movement speed compared with younger animals. Moreover, aged monkeys had significantly reduced ATP synthesis capacity (in substantia nigra and PUT), reduced pyruvate dehydrogenase activity (in PUT), and reduced calcium buffering capacity (in PUT) compared with younger animals. Furthermore, this age-related decline in mitochondrial function in the basal ganglia correlated with decline in motor function. Overall, our results suggest that drug therapies designed to enhance altered mitochondrial function may help improve motor deficits in the elderly.