Anand N. Rao

Stability properties of neuronal microtubules.

Neurons are terminally differentiated cells that use their microtubule arrays not for cell division but rather as architectural elements required for the elaboration of elongated axons and dendrites. In addition to acting as compression‐bearing struts that provide for the shape of the neuron, microtubules also act as directional railways for organelle transport. The stability properties of neuronal microtubules are commonly discussed in the biomedical literature as crucial to the development and maintenance of the nervous system, and have recently gained attention as central to the etiology of neurodegenerative diseases. Drugs that affect microtubule stability are currently under investigation as potential therapies for disease and injury of the nervous system. There is often a lack of consistency, however, in how the issue of microtubule stability is discussed in the literature, and this can affect the design and interpretation of experiments as well as potential therapeutic regimens. Neuronal microtubules are considered to be more stable than microtubules in dividing cells. On average, this is true, but in addition to an abundant stable microtubule fraction in neurons, there is also an abundant labile microtubule fraction. Both are functionally important. Individual microtubules consist of domains that differ in their stability properties, and these domains can also differ markedly in their composition as well as how they interact with various microtubule‐related proteins in the neuron. Myriad proteins and pathways have been discussed as potential contributors to microtubule stability in neurons.

The Antiparkinsonian and Antidyskinetic Mechanisms of Mucuna pruriens in the MPTP-Treated Nonhuman Primate

Chronic treatment with levodopa (LD) in Parkinsons disease (PD) can cause drug induced dyskinesias. Mucuna pruriens endocarp powder (MPEP) contains several compounds including natural LD and has been reported to not cause drug-induced dyskinesias. We evaluated the effects of Mucuna pruriens to determine if its underlying mechanistic actions are exclusively due to LD. We first compared MPEP with and without carbidopa (CD), and LD+CD in hemiparkinsonian (HP) monkeys. Each treatment ameliorated parkinsonism. We then compared the neuronal firing properties of the substantia nigra reticulata (SNR) and subthalamic nucleus (STN) in HP monkeys with MPEP+CD and LD+CD to evaluate basal ganglia circuitry alterations. Both treatments decreased SNR firing rate compared to HP state. However, LD+CD treatments significantly increased SNR bursting firing patterns that were not seen with MPEP+CD treatments. No significant changes were seen in STN firing properties. We then evaluated the effects of a water extract of MPEP. Oral MPWE ameliorated parkinsonism without causing drug-induced dyskinesias. The distinctive neurophysiological findings in the basal ganglia and the ability to ameliorate parkinsonism without causing dyskinesias strongly suggest that Mucuna pruriens acts through a novel mechanism that is different from that of LD.

The effect of striatal dopaminergic grafts on the neuronal activity in the substantia nigra pars reticulata and subthalamic nucleus in hemiparkinsonian rats

The electrophysiological correlates of parkinsonism in the basal ganglia have been well studied in patients with Parkinsons disease and animal models. Separately, striatal dopaminergic cell transplantation has shown promise in ameliorating parkinsonian motor symptoms. However, the effect of dopaminergic grafts on basal ganglia electrophysiology has not thoroughly been investigated. In this study, we transplanted murine foetal ventral mesencephalic cells into rats rendered hemiparkinsonian by 6-hydroxydopamine injection. Three months after transplantation, extracellular and local field potential recordings were taken under urethane anaesthesia from the substantia nigra pars reticulata and subthalamic nucleus along with cortical electroencephalograms and were compared to recordings from normal and hemiparkinsonian controls. Recordings from cortical slow-wave activity and global activation states were analysed separately. Rats with histologically confirmed xenografts showed behavioural improvement measured by counting apomorphine-induced rotations and with the extended body axis test. Firing rates in both nuclei were not significantly different between control and grafted groups. However, burst firing patterns in both nuclei in the slow-wave activity state were significantly reduced (P < 0.05) in rats with large surviving grafts, compared to hemiparkinsonian controls. The neuronal firing entropies and oscillations in both nuclei were restored to normal levels in the large-graft group. Electroencephalogram spike-triggered averages also showed normalization in the slow-wave activity state (P < 0.05). These results suggest that local continuous dopaminergic stimulation exerts a normalizing effect on the downstream parkinsonian basal ganglia firing patterns. This novel finding is relevant to future preclinical and clinical investigations of cell transplantation and the development of next-generation therapies for Parkinsons disease that ameliorate pathophysiological neural activity and provide optimal recovery of function.

Cytoplasmic Dynein Transports Axonal Microtubules in a Polarity-Sorting Manner

Axonal microtubules are predominantly organized into a plus-end-out pattern. Here, we tested both experimentally and with computational modeling whether a motor-based polarity-sorting mechanism can explain this microtubule pattern. The posited mechanism centers on cytoplasmic dynein transporting plus-end-out and minus-end-out microtubules into and out of the axon, respectively. When cytoplasmic dynein was acutely inhibited, the bi-directional transport of microtubules in the axon was disrupted in both directions, after which minus-end-out microtubules accumulated in the axon over time. Computational modeling revealed that dynein-mediated transport of microtubules can establish and preserve a predominantly plus-end-out microtubule pattern as per the details of the experimental findings, but only if a kinesin motor and a static cross-linker protein are also at play. Consistent with the predictions of the model, partial depletion of TRIM46, a protein that cross-links axonal microtubules in a manner that influences their polarity orientation, leads to an increase in microtubule transport.

Sliding of centrosome-unattached microtubules defines key features of neuronal phenotype

Rao et al. show that during migration, neurons contain a small population of centrosome-unattached microtubules in the leading process that are capable of sliding. Increasing the proportion of centrosome-unattached microtubules alters neuronal morphology, migration path, and microtubule behavior in the leading process.

Pharmacologically increasing microtubule acetylation corrects stress-exacerbated effects of organophosphates on neurons

Many veterans of the 1990‐1991 Gulf War contracted Gulf War Illness (GWI), a multisymptom disease that primarily affects the nervous system. Here, we treated cultures of human or rat neurons with diisopropyl fluorophosphate (DFP), an analog of sarin, one of the organophosphate (OP) toxicants to which the military veterans were exposed. All observed cellular defects produced by DFP were exacerbated by pretreatment with corticosterone or cortisol, which, in rat and human neurons, respectively, serves in our experiments to mimic the physical stress endured by soldiers during the war. To best mimic the disease, DFP was used below the level needed to inhibit acetylcholinesterase. We observed a diminution in the ratio of acetylated to total tubulin that was correctable by treatment with tubacin, a drug that inhibits HDAC6, the tubulin deacetylase. The reduction in microtubule acetylation was coupled with deficits in microtubule dynamics, which were correctable by HDAC6 inhibition. Deficits in mitochondrial transport and dopamine release were also improved by tubacin. Thus, various negative effects of the toxicant/stress exposures were at least partially correctable by restoring microtubule acetylation to a more normal status. Such an approach may have therapeutic benefit for individuals suffering from GWI or other neurological disorders linked to OP exposure.

Polarity Sorting of Microtubules in the Axon

A longstanding question in cellular neuroscience is how microtubules in the axon become organized with their plus ends out, a pattern starkly different from the mixed orientation of microtubules in vertebrate dendrites. Recent attention has focused on a mechanism called polarity sorting, in which microtubules of opposite orientation are spatially separated by molecular motor proteins. Here we discuss this mechanism, and conclude that microtubules are polarity sorted in the axon by cytoplasmic dynein but that additional factors are also needed. In particular, computational modeling and experimental evidence suggest that static crosslinking proteins are required to appropriately restrict microtubule movements so that polarity sorting by cytoplasmic dynein can occur in a manner unimpeded by other motor proteins.

Truncating mutations of SPAST associated with hereditary spastic paraplegia indicate greater accumulation and toxicity of the M1 isoform of spastin

The SPAST gene, which produces two isoforms of the microtubule-severing protein spastin, is the chief gene mutated in hereditary spastic paraplegia. Truncated M1 spastin proteins are toxic and have the potential to accumulate in these patients.

Polarity sorting of axonal microtubules: a computational study

An essential feature of healthy axons is the development of a uniform microtubule (MT) polarity pattern. Computational simulations support a model in which MTs are polarity sorted in the axon by dynein-based gliding, occasionally opposed by plus-directed motors. We predict that cross-linking proteins limit the mobility of longer MTs.

Transfer entropy between cortical and basal ganglia electrophysiology

Linear measures such as cross-correlation, coherence, and directed transfer functions have previously been applied to investigate the functional connectivity between brain regions. However, such methods do not account for nonlinear interactions between the signals. Separately, dopaminergic cell transplants have been shown to provide symptomatic amelioration and partial electrophysiological normalization of aberrant basal ganglia firing patterns in Parkinsons Disease. However, the precise extent and mechanisms of basal ganglia electrophysiological normalization have remained unclear. In this experiment we computed the transfer entropy between electroencephalograms (EEGs) and basal ganglia local field potentials (LFPs) from urethane-anesthetized rats, in order to investigate both linear and nonlinear interactions. We used the 6-hydroxy-dopamine lesioned medial forebrain bundle hemiparkinsonian (HP) rat model, and recorded from the substantia nigra and subthalamic nucleus of normal rats, HP rats, and HP rats with murine fetal ventral mesencephalic cell transplants, looking separately at slow wave EEG epochs versus global activation epochs. We found that both the crosscorrelation and the transfer entropy between the motor cortical EEG and basal ganglia LFPs was increased in the HP group (p<;0.05) and returned to normal levels in the grafted group, in most nuclei and conditions. However, the transfer entropy more robustly showed the difference between the groups. Our findings indicate that transfer entropy is a sensitive tool for nonlinear inter-nucleic functional connectivity analyses, and demonstrate the novel restorative ability of dopaminergic grafts for the parkinsonian basal ganglia electrophysiology.