Improved Behavioral Response as a Valid Biomarker for Drug Screening Program in Transgenic Rodent Models of Tauopathies

Neurodegenerative tauopathies are defined as a group of dementia and movement disorders characterized by prominent filamentous tau inclusions and degeneration located within certain brain regions. Their common sign is a presence of proteinaceous aggregates composed of hyperphosphorylated and truncated tau proteins. The molecular mechanisms of the disease still remain unresolved, therefore transgenic organisms displaying tau-related neurodegenerative cascade have been created to allow decoding of individual pathways involved in human pathological conditions. Moreover, use of transgenic model organisms enables the application of potential therapeutic approaches. The expression of mutated or misfolded tau as a transgene in vivo leads to significant alteration of neurobehavioral features of experimental animal, therefore detailed classification of behavioral phenotype become one of the first crucial analyses, while it functionally correlates with central nervous system impairment. Currently, two major types of behavioral impairment have been described in transgenic rodent models of tauopathies, (1) progressive motor impairment associated with muscular weakness and premature death and (2) age-related impairment of cognitive functions attended with unaffected motor status. Up to the present, only transgenic models displaying motor impairment were successfully applied into the drug trials targeting misfolded tau protein, despite their behavioral inconsistence with clinical profile of progressive human tauopathy. The aim of this study was, therefore, to summarize the pros and cons of used transgenic rodent models mimicking human tauopathies in connection with development of therapeutic strategies.     

Transgenic mouse models of spinal and bulbar muscular atrophy (SBMA)

Spinal and bulbar muscular atrophy (SBMA) is a late-onset motor neuron disease characterized by proximal muscle atrophy, weakness, contraction fasciculations, and bulbar involvement. Only males develop symptoms, while female carriers usually are asymptomatic. A specific treatment for SBMA has not been established. The molecular basis of SBMA is the expansion of a trinucleotide CAG repeat, which encodes the polyglutamine (polyQ) tract, in the first exon of the androgen receptor (AR) gene. The pathologic hallmark is nuclear inclusions (NIs) containing the mutant and truncated AR with expanded polyQ in the residual motor neurons in the brainstem and spinal cord as well as in some other visceral organs. Several transgenic (Tg) mouse models have been created for studying the pathogenesis of SBMA. The Tg mouse model carrying pure 239 CAGs under human AR promoter and another model carrying truncated AR with expanded CAGs show motor impairment and nuclear NIs in spinal motor neurons. Interestingly, Tg mice carrying full-length human AR with expanded polyQ demonstrate progressive motor impairment and neurogenic pathology as well as sexual difference of phenotypes. These models recapitulate the phenotypic expression observed in SBMA. The ligand-dependent nuclear localization of the mutant AR is found to be involved in the disease mechanism, and hormonal therapy is suggested to be a therapeutic approach applicable to SBMA.     

Experimental Muscle Pain Impairs the Synergistic Modular Control of Neck Muscles

A motor task can be performed via different patterns of muscle activation that show regularities that can be factorized in combinations of a reduced number of muscle groupings (also referred to as motor modules, or muscle synergies). In this study we evaluate whether an acute noxious stimulus induces a change in the way motor modules are combined to generate movement by neck muscles. The neck region was selected as it is a region with potentially high muscular redundancy. We used the motor modules framework to assess the redistribution of muscular activity of 12 muscles (6 per side) in the neck region of 8 healthy individuals engaged in a head and neck aiming task, in non-painful conditions (baseline, isotonic saline injection, post pain) and after the injection of hypertonic saline into the right splenius capitis muscle. The kinematics of the task was similar in the painful and control conditions. A general decrease of activity was noted for the injected muscle during the painful condition together with an increase or decrease of the activity of the other muscles. Subjects did not adopt shared control strategies (motor modules inter subject similarity at baseline 0.73±0.14); the motor modules recorded during the painful condition could not be used to reconstruct the activation patterns of the control conditions, and the painful stimulus triggered a subject-specific redistribution of muscular activation (i.e., in some subjects the activity of a given muscle increased, whereas in other subjects it decreased with pain). Alterations of afferent input (i.e., painful stimulus) influenced motor control at a multi muscular level, but not kinematic output. These findings provide new insights into the motor adaptation to pain.     

Leuprorelin rescues polyglutamine-dependent phenotypes in a transgenic mouse model of spinal and bulbar muscular atrophy

Spinal and bulbar muscular atrophy (SBMA) is an adult-onset motor neuron disease that affects males. It is caused by the expansion of a polyglutamine (polyQ) tract in androgen receptors. Female carriers are usually asymptomatic. No specific treatment has been established. Our transgenic mouse model carrying a full-length human androgen receptor with expanded polyQ has considerable gender-related motor impairment. This phenotype was abrogated by castration, which prevented nuclear translocation of mutant androgen receptors. We examined the effect of androgen-blockade drugs on our mouse model. Leuprorelin, a lutenizing hormone-releasing hormone (LHRH) agonist that reduces testosterone release from the testis, rescued motor dysfunction and nuclear accumulation of mutant androgen receptors in male transgenic mice. Moreover, leuprorelin treatment reversed the behavioral and histopathological phenotypes that were once caused by transient increases in serum testosterone. Flutamide, an androgen antagonist promoting nuclear translocation of androgen receptors, yielded no therapeutic effect. Leuprorelin thus seems to be a promising candidate for the treatment of SBMA.     

Selective Vulnerability of Spinal and Cortical Motor Neuron Subpopulations in delta7 SMA Mice

Loss of the survival motor neuron gene (SMN1) is responsible for spinal muscular atrophy (SMA), the most common inherited cause of infant mortality. Even though the SMA phenotype is traditionally considered as related to spinal motor neuron loss, it remains debated whether the specific targeting of motor neurons could represent the best therapeutic option for the disease. We here investigated, using stereological quantification methods, the spinal cord and cerebral motor cortex of ∆7 SMA mice during development, to verify extent and selectivity of motor neuron loss. We found progressive post-natal loss of spinal motor neurons, already at pre-symptomatic stages, and a higher vulnerability of motor neurons innervating proximal and axial muscles. Larger motor neurons decreased in the course of disease, either for selective loss or specific developmental impairment. We also found a selective reduction of layer V pyramidal neurons associated with layer V gliosis in the cerebral motor cortex. Our data indicate that in the ∆7 SMA model SMN loss is critical for the spinal cord, particularly for specific motor neuron pools. Neuronal loss, however, is not selective for lower motor neurons. These data further suggest that SMA pathogenesis is likely more complex than previously anticipated. The better knowledge of SMA models might be instrumental in shaping better therapeutic options for affected patients.     

Mouse models of SMA: tools for disease characterization and therapeutic development

Mouse models of human disease are an important tool for studying disease mechanism and manifestation in a way that is physiologically relevant. Spinal muscular atrophy (SMA) is a neurodegenerative disease that is caused by deletion or mutation of the survival motor neuron gene (SMN1). The SMA disease is present in a spectrum of disease severities ranging from infant mortality, in the most severe cases, to minor motor impairment, in the mildest cases. The variability of disease severity inversely correlates with the copy number, and thus expression of a second, partially functional survival motor neuron gene, SMN2. Correspondingly, a plethora of mouse models has been developed to mimic these different types of SMA. These models express a range of SMN protein levels and extensively cover the severe and mild types of SMA, with neurological and physiological manifestation of disease supporting the relevance of these models. The SMA models provide a strong background for studying SMA and have already shown to be useful in pre-clinical therapeutic studies. The purpose of this review is to succinctly summarize the genetic and disease characteristic of the SMA mouse models and to highlight their use for therapeutic testing.     

Increased Muscle Activation Following Motor Imagery During the Rehabilitation of the Anterior Cruciate Ligament

Motor imagery (MI) is the mental representation of an action without any concomitant movement. MI has been used frequently after peripheral injuries to decrease pain and facilitate rehabilitation. However, little is known about the effects of MI on muscle activation underlying the motor recovery. This study aimed to assess the therapeutic effects of MI on the activation of lower limb muscles, as well as on the time course of functional recovery and pain after surgery of the anterior cruciate ligament (ACL). Twelve patients with a torn ACL were randomly assigned to a MI or control group, who both received a series of physiotherapy. Electromyographic activity of the quadriceps, pain, anthropometrical data, and lower limb motor ability were measured throughout a 12-session therapy. The data provided evidence that MI elicited greater muscle activation, even though imagery practice did not result in pain decrease. Muscle activation increase might originate from a redistribution of the central neuronal activity, as there was no anthropometric change in lower limb muscles after imagery practice. This study confirmed the effectiveness of integrating MI in a rehabilitation process by facilitating muscular properties recovery following motor impairment. MI may thus be considered a reliable adjunct therapy to help injured patients to recover motor functions after reconstructive surgery of ACL.     

The effects of verapamil and ethanol on body temperature and motor coordination

Male, adult mice of the Binghamton heterogeneous stock received one of two doses of ethanol (1.0 g/kg or 2.0 g/kg in saline) alone or in combination with the calcium (Ca2+) slow channel blocker, verapamil (5.45 mg/kg in 25% v/v ethanol in saline). Hypothermic responses and motor incoordination were assessed in terms of rectal temperatures and rotorod activity both 20 and 60 min after drug administration. Verapamil alone did not affect body temperature, but it potentiated ethanol-induced hypothermia at both post-administration test times. Both verapamil and ethanol impaired muscular coordination and these effects were additive at the two observation periods. Verapamil did not affect ethanol blood levels from 10 to 80 min after administration of the drugs. Since motor impairment was observed when verapamil was administered with only its ethanol vehicle, this suggests a powerful interactive effect between the two drugs.     

Olfactory functions at the intersection between environmental exposure to manganese and Parkinsonism

The olfactory function can be affected by occupational and environmental exposure to various neurotoxicants that can be transported through the olfactory pathway. Olfactory impairment is a highly recurrent non-motor dysfunction in Parkinson's disease and is considered an early predictive sign of neurodegeneration. Changes in olfactory perception may be caused by a dopaminergic dysregulation, possibly related to changes at the level of dopamine receptors. Manganese is an essential element that can become neurotoxic in various conditions inducing an overload in the organism. Being actively transported through the olfactory tract, manganese can cause impairment of olfactory function and motor coordination in different age groups like children and elderly. Odor and motor changes are interrelated and may be caused by a Mn-induced dopaminergic dysregulation affecting both functions. Given these findings, further research is imperative on the possible role of manganese exposure as a pathogenetic factor for Parkinsonism.     

Model-based development of neuroprosthesis for paraplegic patients.

In paraplegic patients with upper motor neuron lesions the signal path from the central nervous system to the muscles is interrupted. Functional electrical stimulation applied to the lower motor neurons can replace the lacking signals. A so-called neuroprosthesis may be used to restore motor function in paraplegic patients on the basis of functional electrical stimulation. However, the control of multiple joints is difficult due to the complexity, nonlinearity, and time-variance of the system involved. Furthermore, effects such as muscle fatigue, spasticity, and limited force in the stimulated muscle further complicate the control task. Mathematical models of the human musculoskeletal system can support the development of neuroprosthesis. In this article a detailed overview of the existing work in the literature is given and two examples developed by the author are presented that give an insight into model-based development of neuroprosthesis for paraplegic patients. It is shown that modelling the musculoskeletal system can provide better understanding of muscular force production and movement coordination principles. Models can also be used to design and test stimulation patterns and feedback control strategies. Additionally, model components can be implemented in a controller to improve control performance. Eventually, the use of musculoskeletal models for neuroprosthesis design may help to avoid internal disturbances such as fatigue and optimize muscular force output. Furthermore, better controller quality can be obtained than in previous empirical approaches. In addition, the number of experimental tests to be performed with human subjects can be reduced. It is concluded that mathematical models play an increasing role in the development of reliable closed-loop controlled, lower extremity neuroprostheses.     

Neuronal adaptation of corticospinal mechanisms of muscle contraction regulation in athletes

Physiological mechanisms of neuronal adaptation of the human corticospinal pathways in response to long-term intense motor activity have been studied insufficiently. In this work, we investigated adaptational changes in corticospinal mechanisms of muscular contraction control in athletes. We measured parameters of motor evoked potentials of lower limb skeletal muscles under voluntary static loads of various intensity and duration, using the transcranial magnetic stimulation method. Elite athletes, as compared to the reference group, in the course of increased intensity and duration of isometric muscular contractions demonstrated more expressed increase in the maximum amplitude of the motor evoked potentials of lower limb skeletal muscles, smaller decrease in the time of central motor conduction of nervous pulses and the peripheral period in electromyograms, and less expressed increase in the cortical and segmental silent periods. Mechanisms of adaptation of corticospinal regulation of human muscular contraction to specific conditions of extreme motor activities are discussed.     

Cognitive cost of motor reorganizations associated with muscular fatigue during a repetitive pointing task

Muscular fatigue is known to impair motor performance and to catalyse the development of upper limb musculoskeletal disorders. In order to delay the deleterious effects of muscular fatigue, the central nervous system (CNS) employs compensatory strategies. The cognitive cost of such compensatory strategies was assessed in 10 male subjects who alternatively performed two dual-task protocols before and immediately after an exhaustion procedure specific to upper arm abductor musculature. The main motor tasks were an isometric force-matching and a rapid multi-joint pointing. A secondary probe reaction time (RT) task was performed during both protocols and served as an indicator of attentional demands. Overall motor task performance was maintained despite fatigue. Kinematic and electromyographic data revealed that subjects used motor reorganization during the pointing task when fatigued. The RT increased with fatigue in both dual-task protocols, but this increase was significantly higher during the pointing task than during the force-matching task.The results highlight that the motor reorganization used by the CNS under muscular fatigue states require higher attentional demands than the initial motor organization. Finally, the capacity to delay the deleterious effects of muscular fatigue seems to depend on the proportion of cognitive resources available to plan the compensatory motor strategy.     

Optical dissection of neural circuits responsible for Drosophila larval locomotion with halorhodopsin

Halorhodopsin (NpHR), a light-driven microbial chloride pump, enables silencing of neuronal function with superb temporal and spatial resolution. Here, we generated a transgenic line of Drosophila that drives expression of NpHR under control of the Gal4/UAS system. Then, we used it to dissect the functional properties of neural circuits that regulate larval peristalsis, a continuous wave of muscular contraction from posterior to anterior segments. We first demonstrate the effectiveness of NpHR by showing that global and continuous NpHR-mediated optical inhibition of motor neurons or sensory feedback neurons induce the same behavioral responses in crawling larvae to those elicited when the function of these neurons are inhibited by Shibire(ts), namely complete paralyses or slowed locomotion, respectively. We then applied transient and/or focused light stimuli to inhibit the activity of motor neurons in a more temporally and spatially restricted manner and studied the effects of the optical inhibition on peristalsis. When a brief light stimulus (1-10 sec) was applied to a crawling larva, the wave of muscular contraction stopped transiently but resumed from the halted position when the light was turned off. Similarly, when a focused light stimulus was applied to inhibit motor neurons in one or a few segments which were about to be activated in a dissected larva undergoing fictive locomotion, the propagation of muscular constriction paused during the light stimulus but resumed from the halted position when the inhibition (>5 sec) was removed. These results suggest that (1) Firing of motor neurons at the forefront of the wave is required for the wave to proceed to more anterior segments, and (2) The information about the phase of the wave, namely which segment is active at a given time, can be memorized in the neural circuits for several seconds.     

Age‐related deterioration of motor function in male and female 5xFAD mice from 3 to 16 months of age

Alzheimer's disease (AD) is a neurodegenerative disorder that leads to age‐related cognitive and sensori‐motor dysfunction. There is an increased understanding that motor dysfunction contributes to overall AD severity, and a need to ameliorate these impairments. The 5xFAD mouse develops the neuropathology, cognitive and motor impairments observed in AD, and thus may be a valuable animal model to study motor deficits in AD. Therefore, we assessed age‐related changes in motor ability of male and female 5xFAD mice from 3 to 16 months of age, using a battery of behavioral tests. At 9‐10 months, 5xFAD mice showed reduced body weight, reduced rearing in the open‐field and impaired performance on the rotarod compared to wild‐type controls. At 12‐13 months, 5xFAD mice showed reduced locomotor activity on the open‐field, and impaired balance on the balance beam. At 15‐16 months, impairments were also seen in grip strength. Although sex differences were observed at specific ages, the development of motor dysfunction was similar in male and female mice. Given the 5xFAD mouse is commonly on a C57BL/6 × SJL hybrid background, a subset of mice may be homozygous recessive for the Dysf ^im mutant allele, which leads to muscular weakness in SJL mice and may exacerbate motor dysfunction. We found small effects of Dysf ^im on motor function, suggesting that Dysf ^im contributes little to motor dysfunction in 5xFAD mice. We conclude that the 5xFAD mouse may be a useful model to study mechanisms that produce motor dysfunction in AD, and to assess the efficacy of therapeutics on ameliorating motor impairment.     

A cell system with targeted disruption of the SMN gene: functional conservation of the SMN protein and dependence of Gemin2 on SMN

The motor neuron degenerative disease spinal muscular atrophy is caused by reduced expression of the survival motor neuron (SMN) protein. Here we report a genetic system developed in the chicken pre-B cell line DT40, in which the endogenous SMN gene is disrupted by homologous recombination, and SMN protein is expressed from a chicken SMN cDNA under control of a tetracycline (tet)-repressible promoter. Addition of tet results in depletion of SMN protein and consequent cell death, which directly demonstrates that SMN is required for cell viability. The tet-induced lethality can be rescued by expression of human SMN, indicating that the function of SMN is highly conserved between the two species. Cells expressing low levels of SMN display slow growth proportional to the amount of SMN they contain. Interestingly, the level of the SMN-interacting protein Gemin2 decreases significantly following depletion of SMN, supporting the conclusion that SMN and Gemin2 form a stable complex in vivo. This system provides a powerful setting for studying the function of SMN in vivo and for screening for potential therapeutics for spinal muscular atrophy.     

Cruciate ligament reflexes.

The idea of muscular reflexes elicited from sensory nerves of the cruciate ligaments is more than 100 years old, but the existence of such reflexes has not been proven until the recent two decades. First in animal experiments, a muscular excitation could be elicited in the hamstrings when the anterior cruciate ligament (ACL) was pulled, and tension in the ligament caused activity of the gamma motor neurones of the muscles around the knee. Impulses from the sensory nerves in ACL were activated during motion of the knee, in particular overstretching and combined extension and rotation. In humans, proprioception in the knee is decreased after ACL rupture. By mechanical or electrical stimulation of the ACL, an excitation in the hamstrings muscles can be elicited. During muscular activity, stimulation of the ACL or PCL results in a clear inhibition of the ongoing activity, both during static isometric and isokinetic muscle work, and also during dynamic activity (gait). This inhibitory reflex subjectively resembled giving way. The latency of the reflex was short in animals (about 3 ms) and long in humans (60-120 ms), probably caused by differences in the experimental setup and between species. The long latency in humans makes it unlikely that it is a directly protective reflex. Instead it may be involved in the updating of motor programs. Further research may characterize the reflex in details and map its pathways. The existence of this reflex indicate that the cruciate ligaments have an afferent function, which influences knee dynamics.     

Family as a factor in cerebral palsy prevention

The aim of the study was to assess maternal perception of family impact on the course and outcome of rehabilitation in children with cerebral motor impairment. The study included 135 children with cerebral motor impairment. Their motor development was followed-up over a one-year period by use of structured interview with the children's mothers after 12-month rehabilitation. The course of rehabilitation was assessed by the method of locomotor system functional evaluation. The improvement achieved in motor development was significantly better in the group of children whose mothers found their relationships with extended family excellent than in those whose mothers considered it good or poor. The study showed that mothers to children with cerebral motor impairment frequently feel the lack of extended family support, being it real or perceived as such by the mothers due to their emotional sensitivity, suggesting the need of additional studies of the reasons for this. These findings indicate that greater attention should be paid by health professionals to the psychological support offered to these mothers.     

Impairment of capping in lymphoblastoid cell lines of Duchenne patients indicates an intrinsic cellular defect

Summary Duchenne muscular dystrophy (DMD) is a lethal sex-linked degenerative disorder of the muscle in man. Generalized cell membrane abnormalities seem to be involved in the pathogenesis of the disease; in particular, the impairment of lymphocyte capping capacities has been repeatedly confimed. To clarify whether capping impairment is a consequence of factors related to the activity of the disease or an expression of an intrinsic cellular defect, we have investigated the capping capacities of DMD EBV-transformed cell lines. The results indicate a significant impairment of capping capacity in cultured cell lines, providing evidence for an intrinsic cell deficiency in DMD.