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| Locomotor and exploratory behavior is preserved in young TDP-43-WT mice. To measure general motor function and exploratory activity we used an open field test. Animals were placed in a novel environment during a 20 min session. (A) Total distance traveled, (B) relative center distance and (C) detailed measurement of total distance traveled in time segments of 5 min. No significant differences were found between controls and bigenic animals in locomotion or exploration (p > 0.05, Student's t test for A,B or repeated-measures ANOVA in C). Number of animals is indicated in parentheses. Data represent mean ± SEM.
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Frontotemporal Dementia (FTD) and amyotrophic lateral sclerosis (ALS) are two neurodegenerative diseases associated to mislocalization and aggregation of TAR DNA-binding protein 43 (TDP-43). To investigate in depth the behavioral phenotype associated with this proteinopathy, we used as a model transgenic (Tg) mice conditionally overexpressing human...
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... motor function and exploratory activity can be evaluated using the open field test. Distance traveled during this task was similar between control and TDP-43-WT groups, suggesting no motor or exploratory abnormalities in our mouse model ( Figure 3A). In addition, relative center distance was also unaffected (Figure 3B). ...
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... traveled during this task was similar between control and TDP-43-WT groups, suggesting no motor or exploratory abnormalities in our mouse model ( Figure 3A). In addition, relative center distance was also unaffected (Figure 3B). To unmask any potential effect on locomotion not revealed by measuring total values, we divided the traveled distance in time bins and confirmed that this parameter was similar between genotypes across the whole duration of the test (Figure 3C). ...
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... addition, relative center distance was also unaffected (Figure 3B). To unmask any potential effect on locomotion not revealed by measuring total values, we divided the traveled distance in time bins and confirmed that this parameter was similar between genotypes across the whole duration of the test (Figure 3C). ...
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... mice displayed a significant increase in the percentage of open arm entries (Student's t test, t (28) = 2.280 p = 0.0304; Figures 5B,C) and a non-significant trend towards increased percentage of time in the open arms (Student's t test, t (28) = 1.459 p = 0.1557, Figure 5D), suggesting decreased anxiety-like behavior compared to the control group. Consistent with the lack of altered locomotion in the open field test (Figures 3A,C), the total number of arm entries and the total distance traveled did not differ between groups (Figures 5E,F). These data argues against an unspecific effect on non-motor behaviors due to increased anxiety or impaired visual function. ...
Citations
... Distance travelled (converted from beam breaks to cm) was recorded at 5-min blocks. 41 The results of open field were not compared with their wild-type counterpart because of the hypermobility associated with TDP-43 transgenic models, 42,43 which creates a different basal for transgenic mice when compared with wild-type mice. ...
Aggregation of the RNA-binding protein TAR DNA binding protein (TDP-43) is a hallmark of TDP-proteinopathies including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). As TDP-43 aggregation and dysregulation are causative of neuronal death, there is a special interest in targeting this protein as a therapeutic approach. Previously, we found that TDP-43 extensively co-aggregated with the dual function protein GEF (guanine exchange factor) and RNA-binding protein rho guanine nucleotide exchange factor (RGNEF) in ALS patients. Here, we show that an N-terminal fragment of RGNEF (NF242) interacts directly with the RNA recognition motifs of TDP-43 competing with RNA and that the IPT/TIG domain of NF242 is essential for this interaction.
Genetic expression of NF242 in a fruit fly ALS model overexpressing TDP-43 suppressed the neuropathological phenotype increasing lifespan, abolishing motor defects and preventing neurodegeneration. Intracerebroventricular injections of AAV9/NF242 in a severe TDP-43 murine model (rNLS8) improved lifespan and motor phenotype, and decreased neuroinflammation markers.
Our results demonstrate an innovative way to target TDP-43 proteinopathies using a protein fragment with a strong affinity for TDP-43 aggregates and a mechanism that includes competition with RNA sequestration, suggesting a promising therapeutic strategy for TDP-43 proteinopathies such as ALS and FTD.
... TDP-43 levels increase with the normal ageing process in humans (Koike et al., 2021), and although TDP-43 pathology has also been reported in many healthy aged individuals (Uchino et al., 2015), the proportion of people with TDP-43 proteinopathy has been reported to be higher in patients with a neurodegenerative condition. Thus, the progressive nature of some cognitive and all motor phenotypes in M323K mutant mice prompted us to look for underlying abnormalities at TDP-43 expression levels, its function and subcellular distribution in brain and spinal cord at different ages (Alfieri et al., 2016;Igaz et al., 2008). Indeed, we found an age-related increase in TDP-43 levels in neuronal tissues of aged wild-type mice, and a more pronounced increase in M323K mutant mice. ...
TDP-43 pathology is found in several neurodegenerative disorders, collectively referred to as “TDP-43 proteinopathies”. Aggregates of TDP-43 are present in the brains and spinal cords of >97% of amyotrophic lateral sclerosis (ALS), and in brains of ∼50% of frontotemporal dementia (FTD) patients. While mutations in the TDP-43 gene (TARDBP) are usually associated with ALS, many clinical reports have linked these mutations to cognitive impairments and/or FTD, but also to other neurodegenerative disorders including Parkinsonism (PD) or progressive supranuclear palsy (PSP). TDP-43 is a ubiquitously expressed, highly conserved RNA-binding protein that is involved in many cellular processes, mainly RNA metabolism. To investigate systemic pathological mechanisms in TDP-43 proteinopathies, aiming to capture the pleiotropic effects of TDP-43 mutations, we have further characterised a mouse model carrying a point mutation (M323K) within the endogenous Tardbp gene. Homozygous mutant mice developed cognitive and behavioural deficits as early as 3 months of age. This was coupled with significant brain structural abnormalities, mainly in the cortex, hippocampus, and white matter fibres, together with progressive cortical interneuron degeneration and neuroinflammation. At the motor level, progressive phenotypes appeared around 6 months of age. Thus, cognitive phenotypes appeared to be of a developmental origin with a mild associated progressive neurodegeneration, while the motor and neuromuscular phenotypes seemed neurodegenerative, underlined by a progressive loss of upper and lower motor neurons as well as distal denervation. This is accompanied by progressive elevated TDP-43 protein and mRNA levels in cortex and spinal cord of homozygous mutant mice from 3 months of age, together with increased cytoplasmic TDP-43 mislocalisation in cortex, hippocampus, hypothalamus, and spinal cord at 12 months of age. In conclusion, we find that Tardbp M323K homozygous mutant mice model many aspects of human TDP-43 proteinopathies, evidencing a dual role for TDP-43 in brain morphogenesis as well as in the maintenance of the motor system, making them an ideal in vivo model system to study the complex biology of TDP-43.
... Researchers have devised a novel transgenic mouse model featuring human wild-type TDP-43 protein (hTDP-43) overexpression, specifically in forebrain neurons. 33,40,41 Initially, Alfieri and colleagues 41 observed the distance traveled and relative center distance in young transgenic hTDP-43 mice during a behavioral task closely resembled those of the control group. This similarity indicated the absence of motor or exploratory abnormalities in the early stages of the ALS model. ...
... For instance, hTDP-43 transgenic mice showed no change in latency to fall in a hang wire test, indicating intact grip strength. 33 However, with advances in technology, the use of digital gauges for quantitative collection and processing of grip strength data has enhanced the reliability of this phenotype as an early indicator of ALS progression. ...
... In contrast, this preference was absent in hTDP-43 transgenic mice, suggesting potential impairment in the functions of the perirhinal and prefrontal cortices. 33 3.2 MORRIS WATER MAZE Like hTDP-43 transgenic mice, mice overexpressing the C9ORF72 mutation exhibit cognitive deficits that can be assessed using the Morris water maze. The Morris water maze consists of a large circular pool or tank filled with opaque water. ...
Amyotrophic lateral sclerosis (ALS) remains an untreatable neurodegenerative disease without a cure or effective treatment, mainly due to elusive underlying mechanisms. ALS is primarily characterized by motor neuron dysfunction in the brain and spinal cord. However, it also exhibits non-motor symptoms such as executive, behavioral, and language dysfunction, making it challenging to establish informative disease models for relevant preclinic and clinical research. The discovery of ALS- causing genes, such as C9orf72 and SOD1, has paved the way for developing various animal models. Among these models, rodents have emerged as particularly valuable, demonstrating unique ALS-related behavioral defects in multiple behavioral tests. These models enable further understanding of disease mechanisms and provide sensitive and precise functional assessments of ALS-related defects for drug development. This report endeavors to present a chronological overview of various behavioral assessments, encompassing motion ability tests, cognitive evaluations, sensory analyses, and other paradigms described in rodent models of ALS. Our goal is to summarize and compare the behavioral alterations observed in diverse rodent models of ALS with distinct gene mutations, thus providing comprehensive references and guidance for advancing pathogenic and therapeutic research in ALS.
... Novel object recognition (NOR) test was performed as described by Alfieri et al. (2016) with minor modifications (Alfieri et al. 2016). Young male and female mice were habituated to an empty Plexiglas arena (40 cm × 23 cm × 15 cm) for 10 min. ...
... Novel object recognition (NOR) test was performed as described by Alfieri et al. (2016) with minor modifications (Alfieri et al. 2016). Young male and female mice were habituated to an empty Plexiglas arena (40 cm × 23 cm × 15 cm) for 10 min. ...
Neonatal exposure to decabromodiphenyl ether (PBDE-209), a widely used flame retardant, affects cognitive performances in the later stage of life in a sex-dependent manner. PBDE-209 interferes with glutamatergic signaling and N-methyl-D-aspartate receptor (NMDAR) subunits with unresolved regulatory mechanisms. This study exposed male and female mice pups through postnatal day (PND) 3-10 to PBDE-209 (oral dose: 0, 6, or 20 mg/kg body weight). The frontal cortex and hippocampus, collected from neonate (PND 11) and young (PND 60) mice, were analyzed for cAMP response element-binding protein (CREB) and RE1-silencing transcription factor/ Neuron-restrictive silencer factor (REST/NRSF) binding to NMDAR1 promoter and expression of NMDAR1 gene by electrophoretic mobility shift assay and semi-quantitative RT-PCR respectively. Behavioral changes were assessed using spontaneous alternation behavior and novel object recognition tests in young mice. In neonates, the binding of CREB was increased, while REST/NRSF was decreased significantly to their cognate NMDAR1 promoter sequences at the high dose of PBDE-209 in both the sexes. This reciprocal pattern of CREB and REST/NRSF interactions correlates with the up-regulation of NMDAR1 expression. Young males followed a similar pattern of CREB and REST/NRSF binding and NMDAR1 expression as in neonates. Surprisingly, young females did not show any alteration when compared to age-matched controls. Also, we found that only young males showed working and recognition memory deficits. These results indicate that early exposure to PBDE-209 interferes with CREB- and REST/NRSF-dependent regulation of the NMDAR1 gene in an acute setting. However, long-term effects persist only in young males that could be associated with cognitive impairment.
Graphical abstract
... Existe evidencia experimental mostrando una reducción en la latencia a caer en modelos de la enfermedad de Parkinson y de esclerosis lateral amiotrófica [23,45]; sin embargo, estos trabajos utilizan solo la latencia a caer como métrica de rendimiento de los animales. Nosotros postulamos que el estudio detallado de los patrones de comportamiento durante la ejecución de tareas nos permitirá la detección temprana de impedimentos motores y la comparación de diferencias entre modelos experimentales de enfermedades neurodegenerativas que ayude a avanzar en la compresión de los circuitos neuronales subyacentes [46]. ...
Animals exhibit complex behavioral repertoires that can be described, with varying degrees of detail, as combinations from a finite set of stereotyped movements or biophysical states.
These biophysical responses are flexible, since different behavior sequences can be used to solve similar tasks, and are adaptable to changing environments through learning mechanisms.
Given this flexibility and adaptability, translating complex animal behaviors into quantifiable features or well-defined behavior categories can be challenging.
On the one hand, manual classification of behaviors can be time-consuming, require the definition of a priori categories and may not be reproducible between assessments.
On the other hand, heuristically created categories (e.g., walking, running or jumping) tend to ignore inherent information regarding intra- and inter-animal variability, frequently found in unrestrained naturalistic settings.
Therefore, in this work we analyze different approaches to quantify and evaluate mouse behavior during the execution of a motor skill learning task (accelerating rotarod). In particular, we apply unsupervised machine learning techniques to classify behaviors (UMAP embeddings with watershed segmentation).
First, we proposed performance metrics, alternative to latency to fall, to showcase varying levels of physical aptitude and task learning amongst mice.
We then used UMAP embeddings to find two possible representations of mouse behavior, in a low dimensional latent space.
One embedding was constructed using mouse bodyparts' wavelet frequency spectra and the other using features extracted from their steps and poses.
Finally, we clustered behaviors into separate categories (labels), by performing watershed segmentation, and characterized them.
In this way, we shed light on the underlying structure and dynamics of behavior, improving our understanding of the execution and learning process of this task. The behaviors found can distinguish between the different mouse performance groups, and the way these behaviors are used is consolidated during training.
... The Y-maze test evaluates rodents short-term memory, locomotors activity and stereotypical behavior (Alfieri et al. 2016). The Y-maze consists of three equally spaced arms labeled with letters A, B and C. Each mouse was placed at the end of one arm for 5 min and allowed to move freely to enter another arm. ...
Alzheimer’s disease (AD) is an irreversible progressive neurodegenerative disorder recognized by accumulation of amyloid-plaques (APs) and neurofibrillary tangles (NFTs) and eventually loss of memory. Glia maturation factor (GMF), a neuroinflammatory protein first time isolated and cloned in our laboratory plays an important role in the pathogenesis of AD. However, no studies have been reported on whether anti-GMF antibody administration could downregulate neuroinflammation and attenuate amyloid pathology in AD brain. We investigated the potential effect of single dose of (2 mg/kg b.wt/mouse) intravenously (iv) injected with anti-GMF antibodyon cognitive function, neuroprotection, neuroinflammation and Aβ load in the brain of 9-month-old 5XFAD mice. Following 4 weeks of anti-GMF antibody delivery in mice, we found reduced expression of GMF, astrocytic glial fibrillary acidic protein (GFAP) and microglial ionizing calcium binding adaptor molecule 1 (Iba1) as well as improvement inneuroinflammatory response via inhibition of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) production and amyloid pathology in the cerebral cortex and hippocampal CA1 region of 5XFAD mice. Correspondingly, blockade of GMF function with anti-GMF antibody improved spatial learning, memory, and long-term recognition memory in 5XFAD mice. The present study demonstrates that the immune checkpoint blockade of GMF function with anti-GMF antibody coordinates anti-inflammatory effects to attenuate neurodegeneration in the cortex and hippocampal CA1 region of 5XFAD mouse brain. Further, our data suggest, that pharmacological immune neutralization of GMF is a promising neuroprotective strategy totherapeutically target neuroinflammation and neurodegeneration in AD.
5XFAD mice Polyclonal anti-GMF antibody
... Existe evidencia experimental mostrando una reducción en la latencia a caer en modelos de la enfermedad de Parkinson y de esclerosis lateral amiotrófica [7,41], sin embargo estos trabajos utilizan solo la latencia de caída como medida del desempeño del animal. Nosotros postulamos que el estudio detallado del patrón de movimiento durante la ejecución de la tarea nos permitirá la detección temprana de impedimentos motores y la comparación de diferencias entre modelos experimentales de enfermedades neurodegenerativas que ayude a avanzar en la compresión de los mecanismos circuitales subyacentes [42]. masa de la celda y el parámetro 0 ≤ θ ≤ 1 permite controlar el grado de aproximación. ...
A fundamental assumption in behavioral neuroscience is that animal activity, while performing defined tasks, can be vastly described by a finite set of stereotyped behaviors. Information about animal behavior can then be correlated with simultaneous recordings of neural activity allowing us to understand how the brain encodes particular behaviors, what are the underlying neural circuits and how these circuits are modified during motor.
However, classifying different types of movements can be a complex endeavor. On the one hand, the extent of animal activity recordings may be too large to be manually classified and such classification may not be reproducible between subjects. On the other, heuristically created categories (e.g., walking, running, jumping) tend to ignore inherent information regarding intra- and inter-animal variability frequently found in unrestrained.
Therefore, in this work we used unsupervised machine learning techniques to classify different types of behaviors exhibited by expert mice performing a motor skill task. In particular, we used the t-SNE algorithm to classify animal behavior from a dataset containing information about the position of the mouse and their body parts while walking on a rotating cylinder at increasing speeds (accelerating rotarod task).
By applying this algorithm, we were able to classify behavior into 9 individual classes that correspond to specific poses of the mouse while performing the accelerating rotarod task. These poses were found to be representative of the individual mice analyzed (N=3) and capture common aspects in their movements. In addition, we studied the dynamics of transitions between poses and their dependence with the rotarod speed. This analysis showed that at faster rotarod speed animals change their motor strategy by selecting a type of locomotion characterized by hindlimb alternation.
Finally, this pose classification was used to study potential correlations with neural activity patterns in the mesencephalic locomotor region (MLR) and we found the existence of individual neurons whose activity is modulated by the occurrence of pose transition events. Altogether, this work demonstrates the benefit of the use of unsupervised strategies for the detailed study of animal behavior and its utility in the study of neural circuit function.
... The Y-maze test evaluates rodent's short-term memory, locomotor activity, and stereotypical behavior. The Y-maze test was conducted as previously described [43]. The Y-maze consists of three equally spaced arms labeled with letters A, B, and C. Each mouse was placed at the end of one arm given 5 min and allowed to move freely to enter another arm. ...
Traumatic brain injury (TBI) induces inflammatory responses through microglial activation and polarization towards a more inflammatory state that contributes to the deleterious secondary brain injury. Glia maturation factor (GMF) is a pro-inflammatory protein that is responsible for neuroinflammation following insult to the brain, such as in TBI. We hypothesized that the absence of GMF in GMF-knockout (GMF-KO) mice would regulate microglial activation state and the M1/M2 phenotypes following TBI. We used the weight drop model of TBI in C57BL/6 mice wild-type (WT) and GMF-KO mice. Immunofluorescence staining, Western blot, and ELISA assays were performed to confirm TBI-induced histopathological and neuroinflammatory changes. Behavioral analysis was done to check motor coordination ability and cognitive function. We demonstrated that the deletion of GMF in GMF-KO mice significantly limited lesion volume, attenuated neuronal loss, inhibited gliosis, and activated microglia adopted predominantly anti-inflammatory (M2) phenotypes. Using an ELISA method, we found a gradual decrease in pro-inflammatory cytokines (TNF-α and IL-6) and upregulation of anti-inflammatory cytokines (IL-4 and IL-10) in GMF-KO mice compared with WT mice, thus, promoting the transition of microglia towards a more predominantly anti-inflammatory (M2) phenotype. GMF-KO mice showed significant improvement in motor ability, memory, and cognition. Overall, our results demonstrate that GMF deficiency regulates microglial polarization, which ameliorates neuronal injury and behavioral impairments following TBI in mice and concludes that GMF is a regulator of neuroinflammation and an ideal therapeutic target for the treatment of TBI.
... The Y-Maze apparatus was used for the forced alternation test of spatial memory and cognitive-like behaviors, similar to previous tests (Alfieri, Silva, & Igaz, 2016 and mice returned to explore all three arms for ten minutes. At the conclusion, mice were returned to their home cage and the apparatus was cleaned with 70% EtOH before running the next test. ...
There is little question about whether astrocytes and TDP-43 are related with neurodegenerative diseases and disorders individually. The research looking at both together, however, is considerably less, and demonstrates conflicting results. Using a cross of two common transgenic mouse models, GFAP-tTA and hTDP-43ΔNLS, to express pathological TDP-43 in astrocytes by forcing TDP-43 to stay in the cytoplasm, the hypotheses are that there will be non-cell autonomous neurodegeneration, as well as behavioral changes, but that there will be no difference related to when the pathological TDP-43 is expressed or differences based on sex. However, results indicated that under the expression of pathological TDP-43, GFAP-tTA/TDP-43ΔNLS mice demonstrate increased anxiety-like behaviors, with females more affected when the pathological TDP-43 is expressed through development (Early Expression) and males more affected when the pathological TDP-43 is suppressed until after wean (Late Expression). In both males and females, under both Early Expression and Late Expression conditions, the astrocytic expression of TDP-43ΔNLS leads to non-cell autonomous neurodegeneration. In vitro primary coculture of cortical neurons and astrocytes also indicates reduced survivability for cells under the GFAP-tTA/TDP-43ΔNLS condition.
... Signs of helplessness, anhedonia, anxiety-like features, cognitive and social abnormalities in FUS-tg animals are characteristic of other FTDL paradigms, that is progranulin-deficient mice, 12 ΔNLS-FUS mice 3 and TDP-43-tg mice. 13 The current study suggests the most prominent FDTL-related deficits of the FUS-tg mice are the classic tests of emotionality, social and hippocampus-dependent performance. In comparison with other FTLD rodent models based on the FUS mutation, the findings reported here in the FUS-tg mice suggest that no hyperactivity or other non-specific behavioural alternations are present, but, typical for this disease, features of anxiety, apathy, cognitive and social deficits are observed. ...
Genetic mutations in FUS, a DNA/RNA‐binding protein, are associated with inherited forms of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). A novel transgenic FUS[1‐359]‐tg mouse line recapitulates core hallmarks of human ALS in the spinal cord, including neuroinflammation and neurodegeneration, ensuing muscle atrophy and paralysis, as well as brain pathomorphological signs of FTLD. However, a question whether FUS[1‐359]‐tg mouse displays behavioural and brain pro‐inflammatory changes characteristic for the FTLD syndrome was not addressed. Here, we studied emotional, social and cognitive behaviours, brain markers of inflammation and plasticity of pre‐symptomatic FUS[1‐359]‐tg male mice, a potential FTLD model. These animals displayed aberrant behaviours and altered brain expression of inflammatory markers and related pathways that are reminiscent to the FTLD‐like syndrome. FTLD‐related behavioural and molecular Journal of Cellular and Molecular Medicine features were studied in the pre‐symptomatic FUS[1‐359]‐tg mice that received standard or new ALS treatments, which have been reported to counteract the ALS‐like syndrome in the mutants. We used anti‐ALS drug riluzole (8 mg/kg/d), or anti‐inflammatory drug, a selective blocker of cyclooxygenase‐2 (celecoxib, 30 mg/kg/d) for 3 weeks, or a single intracerebroventricular (i.c.v.) infusion of human stem cells (Neuro‐Cells, 500 000‐CD34⁺), which showed anti‐inflammatory properties. Signs of elevated anxiety, depressive‐like behaviour, cognitive deficits and abnormal social behaviour were less marked in FUS‐tg–treated animals. Applied treatments have normalized protein expression of interleukin‐1β (IL‐1β) in the prefrontal cortex and the hippocampus, and of Iba‐1 and GSK‐3β in the hippocampus. Thus, the pre‐symptomatic FUS[1‐359]‐tg mice demonstrate FTLD‐like abnormalities that are attenuated by standard and new ALS treatments, including Neuro‐Cell preparation.
