Quantitative Gait Analysis Using a Motorized Treadmill System Sensitively Detects Motor Abnormalities in Mice Expressing ATPase Defective Spastin

The hereditary spastic paraplegias (HSPs) are genetic conditions in which there is progressive axonal degeneration in the corticospinal tract. Autosomal dominant mutations, including nonsense, frameshift and missense changes, in the gene encoding the microtubule severing ATPase spastin are the most common cause of HSP in North America and northern Europe. In this study we report quantitative gait analysis using a motorized treadmill system, carried out on mice knocked-in for a disease-associated mutation affecting a critical residue in the Walker A motif of the spastin ATPase domain. At 4 months and at one year of age homozygous mutant mice had a number of abnormal gait parameters, including in stride length and stride duration, compared to heterozygous and wild-type littermates. Gait parameters in heterozygous animals did not differ from wild-type littermates. We conclude that quantitative gait analysis using the DigiGait system sensitively detects motor abnormalities in a hereditary spastic paraplegia model, and would be a useful method for analyzing the effects of pharmacological treatments for HSP.     

SPG20 protein spartin associates with cardiolipin via its plant-related senescence domain and regulates mitochondrial Ca2+ homeostasis

Hereditary spastic paraplegias (HSPs) are a group of neurological disorders characterized clinically by spasticity of lower limbs and pathologically by degeneration of the corticospinal tract. Troyer syndrome is an autosomal recessive HSP caused by a frameshift mutation in the spartin (SPG20) gene. Previously, we established that this mutation results in a lack of expression of the truncated mutant spartin protein. Spartin is involved in many cellular processes and associates with several intracellular organelles, including mitochondria. Spartin contains a conserved plant-related senescence domain at its C-terminus. However, neither the function of this domain nor the roles of spartin in mitochondrial physiology are currently known. In this study, we determined that the plant-related senescence domain of spartin interacts with cardiolipin but not with two other major mitochondrial phospholipids, phosphatidylcholine and phosphatidylethanolamine. We also found that knockdown of spartin by small interfering RNA in a human neuroblastoma cell line resulted in depolarization of the mitochondrial membrane. In addition, depletion of spartin resulted in a significant decrease in both mitochondrial calcium uptake and mitochondrial membrane potential in cells treated with thapsigargin. Our results suggest that impairment of mitochondrial calcium uptake might contribute to the neurodegeneration of long corticospinal axons and the pathophysiology of Troyer syndrome.     

Interaction between the premotor processes of eye and hand movements: Possible mechanism underlying eye–hand coordination

Interaction between the execution process of eye movement and that of hand movement must be indispensable for eye–hand coordination. The present study investigated corticospinal excitability in the hand muscles during the premotor processes of eye and/or hand movement to elucidate interaction between these processes. Healthy humans performed a precued reaction task of eye and/or finger movement and motor-evoked potentials in the hand muscles were evoked in the reaction time. Leftward eye movement suppressed corticospinal excitability in the right abductor digiti minimi muscle only when little finger abduction was simultaneously executed. Corticospinal excitability in the first dorsal interosseous muscle was not suppressed by eye movement regardless of whether or not it was accompanied by finger movement. Suppression of corticospinal excitability in the hand muscles induced by eye movement in the premotor period depends on the direction of eye movement, the muscle tested, and the premotor process of the tested muscle. The suppression may reflect interaction between the motor process of eye movement and that of hand movement particularly active during eye–hand coordination tasks during which both processes proceed.     

HSPA1A is upregulated in periodontal ligament at early stage of tooth movement in rats

Heat shock proteins (HSPs) are molecular chaperones that maintain intracellular protein homeostasis and ensure survival of cells. Continuous orthodontic force on the tooth is considered to be a type of physical stress loaded to the periodontal ligament (PDL). However, little is known about the role of HSPs during tooth movement. This study was performed to examine the expression of HSPs in the PDL during tooth movement using laser microdissection, microarray analysis, real-time RT-PCR and immunohistochemistry. Gene expression of HSPA1A in the pressure zone of the PDL was higher during 6 h of tooth movement than in the control group. Expression of HSPA1A decreased with time. HSPA1A was also detected in the pressure zone of the PDL at the protein level 24 h after the initial tissue change. These results strongly suggest that expression of HSPA1A in the PDL during early stages of tooth movement is a critical factor for tissue reaction.     

Biodegradable biomatrices and bridging the injured spinal cord: the corticospinal tract as a proof of principle

Important advances in the development of smart biodegradable implants for axonal regeneration after spinal cord injury have recently been reported. These advances are evaluated in this review with special emphasis on the regeneration of the corticospinal tract. The corticospinal tract is often considered the ultimate challenge in demonstrating whether a repair strategy has been successful in the regeneration of the injured mammalian spinal cord. The extensive know-how of factors and cells involved in the development of the corticospinal tract, and the advances made in material science and tissue engineering technology, have provided the foundations for the optimization of the biomatrices needed for repair. Based on the findings summarized in this review, the future development of smart biodegradable bridges for CST regrowth and regeneration in the injured spinal cord is discussed.     

Unilateral practice of a ballistic movement causes bilateral increases in performance and corticospinal excitability.

It has long been known that practicing a task with one limb can result in performance improvements with the opposite, untrained limb. Hypotheses to account for cross-limb transfer of performance state that the effect is mediated either by neural adaptations in higher order control centers that are accessible to both limbs, or that there is a "spillover" of neural drive to the opposite hemisphere that results in bilateral adaptation. Here we address these hypotheses by assessing performance and corticospinal excitability in both hands after unilateral practice of a ballistic finger movement. Participants (n = 9) completed 300 practice trials of a ballistic task with the right hand, the aim of which was to maximize the peak abduction acceleration of the index finger. Practice caused a 140% improvement in right-hand performance and an 82% improvement for the untrained left hand. There were bilateral increases in the amplitude of responses to transcranial magnetic stimulation, but increased corticospinal excitability was not correlated with improved performance. There were no significant changes in corticospinal excitability or task performance for a control group that did not train (n = 9), indicating that performance testing for the left hand alone did not induce performance or corticospinal effects. Although the data do not provide conclusive evidence whether increased corticospinal excitability in the untrained hand is causally related to the cross-transfer of ballistic performance, the finding that ballistic practice can induce bilateral corticospinal adaptations may have important clinical implications for movement rehabilitation.     

Noninvasive stimulation of the human corticospinal tract.

Spinal tracts can be stimulated noninvasively in human subjects by passing a high-voltage stimulus between the mastoids or by magnetic stimulation over the back of the head. The stimulus probably activates the corticospinal tract at the cervicomedullary junction (pyramidal decussation) and evokes large, short-latency motor responses in the arm muscles. These responses have a large monosynaptic component. Responses in leg muscles can be elicited by cervicomedullary junction stimulation or by stimulation over the cervical or thoracic spine. Because nerve roots are more easily activated than spinal tracts, stimulus spread to motor axons can occur. Facilitation of responses by voluntary activity confirms that the responses are evoked synaptically. Stimulation of the corticospinal tract is useful in studies of central conduction and studies of the behavior of motoneurons during different tasks. It also provides an important comparison to allow interpretation of changes in responses to stimulation of the motor cortex. The major drawback to the use of electrical stimulation of the corticospinal tract is that each stimulus is transiently painful.     

Axoplasmic flow of tritiated proline in the corticospinal tract of the rat

Summary The rates of axoplasmic transport were studied in the corticospinal tract of the rat by injecting tritiated proline into the sensory-motor cortex and subsequently analyzing the distribution of incorporated label in the spinal cord at intervals after injection. A mathematical model of the anatomy of the corticospinal tract was developed and used in analysis of the data. The rate of a fast component was calculated to be 240–420 mm per day, which is comparable with rates of fast components in the peripheral nervous system (PNS), but considerably greater than rates in other tracts in the central nervous system. A slow component was calculated to have a transport rate of 3–8 mm per day which is greater than rates found either in the CNS or PNS. This higher rate may be related to the greater length of the corticospinal tract as compared to other CNS tracts studied.This research was financed by the Veterans Administration research support awarded to Dr. Feringa by the Development Funds of the Department of Pathology, University of Michigan, and by the University of Michigan Medical Center Fund for Computing. The authors wish to express their appreciation to Ms. Linda Lee Austin for technical assistance, Ms. Diane Trakas and Ms. Barbara Reader for secretarial aid, and Mr. Richard Fritzler for assistance with graphics     

The effect of eye movement on the control of arm movement to a target

Abstract

The purpose of this study was to investigate the effect of eye movement on the control of arm movement to a target. Healthy humans flexed the elbow to a stationary target in response to a start tone. Simultaneously, the subject moved the eyes to the target (saccade eye movement), visually tracked a laser point moving with the arm (smooth pursuit eye movement), or gazed at a stationary start point at the midline of the horizontal visual angle (non-eye movement—NEM). Arm movement onset was delayed when saccade eye movement accompanied it. The onset of an electromyographic burst in the biceps muscle and the onset of saccade eye movement were almost simultaneous when both the arm and the eyes moved to the target. Arm movement duration during smooth pursuit eye movement was significantly longer than that during saccade eye movement or NEM. In spite of these findings, amplitudes of motor-evoked potential in the biceps and triceps brachii muscles were not significantly different among the eye movement conditions. These findings indicate that eye movement certainly affects the temporal control of arm movement, but may not affect corticospinal excitability in the arm muscles during arm movement.     

A Missense Mutation in SLC33A1, which Encodes the Acetyl-CoA Transporter, Causes Autosomal-Dominant Spastic Paraplegia (SPG42)

Hereditary spastic paraplegias (HSPs), characterized by progressive and bilateral spasticity of the legs, are usually caused by developmental failure or degeneration of motor axons in the corticospinal tract. There are considerable interfamilial and intrafamilial variations in age at onset and severity of spasticity. Genetic studies also showed that there are dozens of genetic loci, on multiple chromosomes, that are responsible for HSPs. Through linkage study of a pedigree of HSP with autosomal-dominant inheritance, we mapped the causative gene to 3q24-q26. Screening of candidate genes revealed that the HSP is caused by a missense mutation in the gene for acetyl-CoA transporter (SLC33A1). It is predicted that the missense mutation, causing the change of the highly conserved serine to arginine at the codon 113 (p. S113R), disrupts the second transmembrane domain in the transporter and reverses the orientation of all of the descending domains. Knockdown of Slc33a1 in zebrafish caused a curve-shaped tail and defective axon outgrowth from the spinal cord. Although the wild-type human SLC33A1 was able to rescue the phenotype caused by Slc33a1 knockdown in zebrafish, the mutant SLC33A1 (p.S113R) was not, suggesting that S113R mutation renders SLC33A1 nonfunctional and one that wild-type allele is not sufficient for sustaining the outgrowth and maintenance of long motor axons in human heterozygotes. Thus, our study illustrated a critical role of acetyl-CoA transporter in motor-neuron development and function.     

Beta-sheet-specific interactions with heat shock proteins define a mechanism of delayed tumor cell death in response to HAMLET

As chaperones, heat shock proteins (HSPs) protect host cells against misfolded proteins that constitute a by-product of protein synthesis. Certain HSPs are also expressed on the surface of tumor cells, possibly to scavenge extracellular unfolded protein ligands and prevent them from becoming cytotoxic. HAMLET—a complex of partially unfolded alpha-lactalbumin and oleic acid—is relying on its N-terminal alpha-helical domain to perturb tumor cell membranes, and the cells die as a consequence of this interaction. Here we show that in parallel, cell surface HSPs bind the beta-sheet domain of alpha-lactalbumin and activate a temporarily protective loop, involving vesicular uptake and lysosomal accumulation. Later, HAMLET destroys lysosomal membrane integrity, and HAMLET release kills the remaining tumor cells. HSPs were identified as HAMLET targets in a proteomic screen and Hsp70-specific antibodies or shRNAs inhibited HAMLET uptake by tumor cells, which showed increased Hsp70 surface expression compared to differentiated cells. The results suggest that HAMLET engages tumor cells by two parallel recognition mechanisms, defined by alpha-helical- or beta-sheet domains of alpha-lactalbumin and resulting in an immediate death response, or a delay due to transient accumulation of the complex in the lysosomes. This dual response pattern was conserved among tumor cells but not seen in normal, differentiated cells. By two different mechanisms, HAMLET thus achieves a remarkably efficient elimination of tumor cells.     

Is Remodelling of Corticospinal Tract Terminations Originating in the Intact Hemisphere Associated with Recovery following Transient Ischaemic Stroke in the Rat?

Following large strokes that encompass the cerebral cortex, it has been suggested that the corticospinal tract originating from the non-ischaemic hemisphere reorganises its pattern of terminal arborisation within the spinal cord to compensate for loss of function. However many strokes in humans predominantly affect subcortical structures with minimal involvement of the cerebral cortex. The aim of the present study was to determine whether remodelling of corticospinal terminals arising from the non-ischaemic hemisphere was associated with spontaneous recovery in rats with subcortical infarcts. Rats were subjected to transient middle cerebral artery occlusion or sham surgery and 28 days later, when animals exhibited functional recovery, cholera toxin b subunit was injected into the contralesional, intact forelimb motor cortex in order to anterogradely label terminals within cervical spinal cord segments. Infarcts were limited to subcortical structures and resulted in partial loss of corticospinal tract axons from the ischaemic hemisphere. Quantitative analysis revealed there was no significant difference in the numbers of terminals on the contralesional side of the spinal grey matter between ischaemic and sham rats. The results indicate that significant remodelling of the corticospinal tract from the non-ischaemic hemisphere is not associated with functional recovery in animals with subcortical infarcts.     

Corticospinal Tract Tracing in the Marmoset with a Clinical Whole-Body 3T Scanner Using Manganese-Enhanced MRI

Manganese-enhanced MRI (MEMRI) has been described as a powerful tool to depict the architecture of neuronal circuits. In this study we investigated the potential use of in vivo MRI detection of manganese for tracing neuronal projections from the primary motor cortex (M1) in healthy marmosets (Callithrix Jacchus). We determined the optimal dose of manganese chloride (MnCl₂) among 800, 400, 40 and 8nmol that led to manganese-induced hyperintensity furthest from the injection site, as specific to the corticospinal tract as possible, and that would not induce motor deficit. A commonly available 3T human clinical MRI scanner and human knee coil were used to follow hyperintensity in the corticospinal tract 24h after injection. A statistical parametric map of seven marmosets injected with the chosen dose, 8 nmol, showed the corticospinal tract and M1 connectivity with the basal ganglia, substantia nigra and thalamus. Safety was determined for the lowest dose that did not induce dexterity and grip strength deficit, and no behavioral effects could be seen in marmosets who received multiple injections of manganese one month apart. In conclusion, our study shows for the first time in marmosets, a reliable and reproducible way to perform longitudinal ME-MRI experiments to observe the integrity of the marmoset corticospinal tract on a clinical 3T MRI scanner.     

Mechanism of hyperventilation in exertion (an uncommon factor)

Electrical stimulation of the corticospinal tract increases the excitability of the phrenic motoneurons directly. As a result the responses of these motoneurons to the impulses from the respiratory centre are facilitated. Until now there were attempts to explain exercise hyperpnea by increasing the activity of the respiratory centre. The experiments permit to suppose that the hyperpnea may be produced by facilitatory action of the corticospinal tract on the respiratory motoneurons, i.e. independently from the respiratory centre.     

A study of the effect of heat shock and metal ions on protein synthesis in Neurospora crassa cells

• 1.1. Neurospora cells were grown at 28°C for 14hr and subjected to heat shock (HS) at 48°C for 45 min. Protein synthesis profiles, monitored by labelling with [³⁵S]methionine and one and two-dimensional electrophoresis, revealed nine heat shock proteins (HSPs).
• 2.2. Crossed-immunoelectrophoresis revealed five polypeptides in the shocked cell extracts that were not detectable in normal cells.
• 3.3. Synthesis of HSPs occurred rapidly during the shock treatment and ceased upon transfer to normal conditions. One of the HSPs—~43 K in size—may be a developmentally-regulated protein.
• 4.4. Metal ions—cadmium, zinc, manganese, copper—did not elicit a stress response when used alone but appeared to modulate the heat shock response.

     

大鼠皮质脊髓束神经电信号的记录与分析方法研究

目的：探索皮质脊髓束（CST）电生理信号的采集记录方法,分析描述电信号的特征,从而为通过植入式微电极阵列进行信号采集的记录方法建立一定的实验基础,为将来进一步研究脊髓损伤修复与功能重建提供有价值的神经电生理基础资料。方法：使用神经信号采集处理系统（Cerebus System）,在SD大鼠的脊髓T8节段处的皮质脊髓束内通过插入微电极,记录大鼠皮质脊髓束神经电信号。利用神经信号分析软件Offline Sorter、Neuroexplorer对已存储的信号文件进行波形特点的描述,包括波长、波幅、放电频率、同一电极上记录到的不同放电单元之间的同步性、两根电极上记录到的不同放电单元之间的同步性、放电信号的峰间期（ISI）分析等。结果：长时间稳定记录到连续的皮质脊髓束自发放电信号,一般在同一电极上记录到3~4个来自不同放电单位（细胞）的放电信号。皮质脊髓束自发放电信号的波形呈双向型,波宽为0.6~1.3 ms,波幅为百μV级。在多次实验状态下均能达到很高的信噪比,信号采集效果理想。经快蓝（LFB）染色确认记录电极尖端位于皮质脊髓束内。结论：本实验采用Cere-bus神经信号采集处理系统,利用记录电极可在大鼠的皮质脊髓束内较长时间稳定地记录到较为稳定的微伏级神经电信号,并可进行有意义的神经电信号特征分析,为进一步研究脊髓损伤修复与功能重建提供了有价值的神经电生理基础资料。     

The corticospinal tract attains a normal configuration in the absence of myelin: observations in jimpy mutant mice

The corticospinal projection was examined in dysmyelinated, jimpy mice and in unaffected littermates following cortical injections of either wheat germ agglutinin conjugated to horseradish peroxidase or biocytin. Corticospinal axons in both phenotypes traverse the medulla within a well-defined pyramidal tract, decussate within several fascicles at the spinomedullary junction, and extend down the spinal cord in a compact bundle in the ventral-most part of the dorsal funiculus. Very few labeled fibers are seen separated from the main bundle. This normal configuration of the corticospinal tract is attained despite the virtual absence of CNS myelin in jimpy mice. It seems unlikely then that the myelin normally present in fiber bundles adjacent to this relatively late emerging projection can significantly influence pathway selection during its development.     

A comparison of corticospinal activation by magnetic coil and electrical stimulation of monkey motor cortex

The effects of different orientations of a Cadwell round magnetic coil (MC) were compared with each other and with surface electrical stimulation of motor cortex in monkeys anesthetized with pentobarbital or urethane. Recordings were made from within the lateral corticospinal tract, either from axonal populations or with a microelectrode from individual axons. A lateral-sagittally orientated MC directly excited corticospinal neurons at lower stimulus intensity than was required for indirect, i.e., transsynaptic excitation via inputs to corticospinal neurons. By contrast, in 2 out of 3 macaques tested, a vertex-tangential orientation could excite corticospinal neurons indirectly at lower intensities than were required for direct excitation; at higher intensities, direct excitation also occurred.The site of direct corticospinal excitation by a lateral-sagitally oriented MC was inferred by comparing the response variability and latency to MC and surface electrical stimuli. Cathodal stimuli elicited more variable corticospinal population responses and later individual axonal responses than were obtained with anodal stimuli. The variability in response is attributed to interaction between nearby, on-going synaptic bombardment and the stimulus, implying that surface cathodal stimuli directly activate corticospinal neurons at the spike trigger zone (presumably the initial segment). By contrast, the consistency and reduced latency of the corticospinal responses to surface anodal stimuli are attributed to the direct excitation of corticospinal fibers within the white matter. When the stimulus intensity is clearly above threshold, surface anodal and cathodal stimuli can activate corticospinal neurons both directly and indirectly.Direct corticospinal excitation by the MC can resemble the effects of either surface anodal or surface cathodal stimuli. We conclude that the MC can activate corticospinal neurons at the spike trigger zone or their fibers deeper in white matter. The findings in the monkey are used to interpret the effects of different MC orientations in the human.     

Nuclear localization and the heat shock proteins

The highly conserved heat shock proteins (HSP) belong to a subset of cellular proteins that localize to the nucleus. HSPs are atypical nuclear proteins in that they localize to the nucleus selectively, rather than invariably. Nuclear localization of HSPs is associated with cell stress and cell growth. This aspect of HSPs is highly conserved with nuclear localization occurring in response to a wide variety of cell stresses. Nuclear localization is likely important for at least some of the heat shock proteins’ protective functions; little is known about the function of the heat shock proteins in the nucleus. Nuclear localization is signalled by the presence of a basic nuclear localization sequence (NLS) within a protein. Though most is known about HSP 72’s nuclear localization, the NLS(s) has not been definitively identified for any of the heat shock proteins. Likely more is involved than presence of a NLS; since the heat shock proteins only localize to the nucleus under selective conditions, nuclear localization must be regulated. HSPs also function as chaperons of nuclear transport, facilitating the movement of other macromolecules across the nuclear membrane. The mechanisms involved in chaperoning of proteins by HSPs into the nucleus are still being identified.