Nimrod Miller

Identification of TMEM230 mutations in familial Parkinson's disease

Parkinsons disease is the second most common neurodegenerative disorder without effective treatment. It is generally sporadic with unknown etiology. However, genetic studies of rare familial forms have led to the identification of mutations in several genes, which are linked to typical Parkinsons disease or parkinsonian disorders. The pathogenesis of Parkinsons disease remains largely elusive. Here we report a locus for autosomal dominant, clinically typical and Lewy body–confirmed Parkinsons disease on the short arm of chromosome 20 (20pter-p12) and identify TMEM230 as the disease-causing gene. We show that TMEM230 encodes a transmembrane protein of secretory/recycling vesicles, including synaptic vesicles in neurons. Disease-linked TMEM230 mutants impair synaptic vesicle trafficking. Our data provide genetic evidence that a mutant transmembrane protein of synaptic vesicles in neurons is etiologically linked to Parkinsons disease, with implications for understanding the pathogenic mechanism of Parkinsons disease and for developing rational therapies.

SnapShot-Seq: A Method for Extracting Genome-Wide, In Vivo mRNA Dynamics from a Single Total RNA Sample

mRNA synthesis, processing, and destruction involve a complex series of molecular steps that are incompletely understood. Because the RNA intermediates in each of these steps have finite lifetimes, extensive mechanistic and dynamical information is encoded in total cellular RNA. Here we report the development of SnapShot-Seq, a set of computational methods that allow the determination of in vivo rates of pre-mRNA synthesis, splicing, intron degradation, and mRNA decay from a single RNA-Seq snapshot of total cellular RNA. SnapShot-Seq can detect in vivo changes in the rates of specific steps of splicing, and it provides genome-wide estimates of pre-mRNA synthesis rates comparable to those obtained via labeling of newly synthesized RNA. We used SnapShot-Seq to investigate the origins of the intrinsic bimodality of metazoan gene expression levels, and our results suggest that this bimodality is partly due to spillover of transcriptional activation from highly expressed genes to their poorly expressed neighbors. SnapShot-Seq dramatically expands the information obtainable from a standard RNA-Seq experiment.

Motor Neuron Mitochondrial Dysfunction in Spinal Muscular Atrophy

Spinal muscular atrophy (SMA), the leading genetic cause of infant mortality, predominantly affects high metabolic tissues including motor neurons, skeletal muscles and the heart. Although the genetic cause of SMA has been identified, mechanisms underlying tissue-specific vulnerability are not well understood. To study these mechanisms, we carried out a deep sequencing analysis of the transcriptome of spinal motor neurons in an SMA mouse model, in which we unexpectedly found changes in many genes associated with mitochondrial bioenergetics. Importantly, functional measurement of mitochondrial activities showed decreased basal and maximal mitochondrial respiration in motor neurons from SMA mice. Using a reduction-oxidation sensitive GFP and fluorescence sensors specifically targeted to mitochondria, we found increased oxidative stress level and impaired mitochondrial membrane potential in motor neurons affected by SMA. In addition, mitochondrial mobility was impaired in SMA disease conditions, with decreased retrograde transport but no effect on anterograde transport. We also found significantly increased fragmentation of the mitochondrial network in primary motor neurons from SMA mice, with no change in mitochondria density. Electron microscopy study of SMA mouse spinal cord revealed mitochondria fragmentation, edema and concentric lamellar inclusions in motor neurons affected by the disease. Intriguingly, these functional and structural deficiencies in the SMA mouse model occur during the presymptomatic stage of disease, suggesting a role in initiating SMA. Altogether, our findings reveal a critical role for mitochondrial defects in SMA pathogenesis and suggest a novel target for improving tissue health in the disease.

Non-Aggregating Tau Phosphorylation by Cyclin-Dependent Kinase 5 Contributes to Motor Neuron Degeneration in Spinal Muscular Atrophy

Mechanisms underlying motor neuron degeneration in spinal muscular atrophy (SMA), the leading inherited cause of infant mortality, remain largely unknown. Many studies have established the importance of hyperphosphorylation of the microtubule-associated protein tau in various neurodegenerative disorders, including Alzheimers and Parkinsons diseases. However, tau phosphorylation in SMA pathogenesis has yet to be investigated. Here we show that tau phosphorylation on serine 202 (S202) and threonine 205 (T205) is increased significantly in SMA motor neurons using two SMA mouse models and human SMA patient spinal cord samples. Interestingly, phosphorylated tau does not form aggregates in motor neurons or neuromuscular junctions (NMJs), even at late stages of SMA disease, distinguishing it from other tauopathies. Hyperphosphorylation of tau on S202 and T205 is mediated by cyclin-dependent kinase 5 (Cdk5) in SMA disease condition, because tau phosphorylation at these sites is significantly reduced in Cdk5 knock-out mice; genetic knock-out of Cdk5 activating subunit p35 in an SMA mouse model also leads to reduced tau phosphorylation on S202 and T205 in the SMA;p35−/− compound mutant mice. In addition, expression of the phosphorylation-deficient tauS202A,T205A mutant alleviates motor neuron defects in a zebrafish SMA model in vivo and mouse motor neuron degeneration in culture, whereas expression of phosphorylation-mimetic tauS202E,T205E promotes motor neuron defects. More importantly, genetic knock-out of tau in SMA mice rescues synapse stripping on motor neurons, NMJ denervation, and motor neuron degeneration in vivo. Altogether, our findings suggest a novel mechanism for SMA pathogenesis in which hyperphosphorylation of non-aggregating tau by Cdk5 contributes to motor neuron degeneration.

Nitric oxide and histamine signal attempts to swallow: A component of learning that food is inedible in Aplysia

Memory that food is inedible in Aplysia arises from training requiring three contingent events. Nitric oxide (NO) and histamine are released by a neuron responding to one of these events, attempts to swallow food. Since NO release during training is necessary for subsequent memory and NO substitutes for attempts to swallow, it was suggested that NO functions during training as a signal of attempts to swallow. However, it has been shown that NO may also be released in other contexts affecting feeding, raising the possibility that its role in learning is unrelated to signaling attempts to swallow. We confirmed that NO during learning signals attempts to swallow, by showing that a variety of behavioral effects on feeding of blocking or adding NO do not affect learning and memory that a food is inedible. In addition, histamine had effects similar to NO on learning that food is inedible, as expected if the transmitters are released together when animals attempt to swallow. Blocking histamine during training blocked long-term memory, and exogenous histamine substituted for attempts to swallow. NO also substituted for histamine during training. Histamine at concentrations relevant to learning activates neuron metacerebral cell (MCC). However, MCC activity is not a good monitor of attempts to swallow during training, since the neuron responds equally well to other stimuli. These findings support and extend the hypothesis that NO and histamine signal efforts to swallow during learning, acting on targets other than the MCC that specifically respond to attempts to swallow.

Impaired Autophagy and Defective Mitochondrial Function: Converging Paths on the Road to Motor Neuron Degeneration

Selective motor neuron degeneration is a hallmark of amyotrophic lateral sclerosis (ALS). Around 10% of all cases present as familial ALS (FALS), while sporadic ALS (SALS) accounts for the remaining 90%. Diverse genetic mutations leading to FALS have been identified, but the underlying causes of SALS remain largely unknown. Despite the heterogeneous and incompletely understood etiology, different types of ALS exhibit overlapping pathology and common phenotypes, including protein aggregation and mitochondrial deficiencies. Here, we review the current understanding of mechanisms leading to motor neuron degeneration in ALS as they pertain to disrupted cellular clearance pathways, ATP biogenesis, calcium buffering and mitochondrial dynamics. Through focusing on impaired autophagic and mitochondrial functions, we highlight how the convergence of diverse cellular processes and pathways contributes to common pathology in motor neuron degeneration.

l-arginine via nitric oxide is an inhibitory feedback modulator of Aplysia feeding

An increase in L-arginine hemolymph concentration acts as a postingestion signal inhibiting Aplysia feeding. At physiological concentrations (a 10-μM increase over background), the inhibitory effect of L-arginine is too weak to block feeding in hungry animals. However, a 10-μM increase in L-arginine concentration acts along with another inhibitory stimulus, the sustained presence of food odor, to inhibit feeding after a period of access to food. A physiological concentration of L-arginine also blocked the excitatory effect of a stimulus enhancing feeding, pheromones secreted by mating conspecifics. High concentrations of L-arginine (2.5 mM) alone also inhibited ad libitum feeding. L-arginine is the substrate from which nitric oxide synthase (NOS) produces nitric oxide (NO). Both an NO donor and a 10-μM increase in L-arginine inhibited biting in response to a weak food stimulus. Treatment with NOS inhibitors initiated food-finding and biting in the absence of food, indicating that food initiates feeding against a background of tonic nitrergic inhibition. Increased feeding in response to blocking NOS is accompanied by firing of the metacerebral (MCC) neuron, a monitor of food arousal. The excitatory effect on the MCC of blocking NOS is indirect. The data suggest that L-arginine acts by amplifying NO synthesis, which acts as a background stimulus inhibiting feeding. Background modulation of neural activity and behavior by NO may also be present in other systems, but such modulation may be difficult to identify because its effects are evident only in the context of additional stimuli modulating behavior.

Nitric oxide induces aspects of egg-laying behavior in Aplysia.

SUMMARY Aplysia egg laying is a complex behavior requiring synchronized activity in many organs. Aspects of the behavior are synchronized via the direct effects of peptide bag cell neurohormones and via stimuli arising during the behavior. Stimuli synchronizing egg laying were examined by treating A. fasciata with a nitric oxide (NO) donor. NO elicited normal appetitive and consummatory behaviors leading to the deposition of cordons containing egg capsules without eggs. The sites at which NO acts were investigated. The latency to egg deposition in response to a NO donor was shorter than that in response to other stimuli, consistent with NO acting at downstream sites from those affected by the other stimuli. The NO donor does not act on neurons in the head ganglia presynaptic to the bag cells or on the bag cells. Ligating the small hermaphroditic duct connecting the gonad to the accessory genital mass blocked egg laying in response to bag cell homogenates, but not in response to exogenous NO, indicating that NO does not act on the gonad. NO is released by transport of eggs along the small hermaphroditic duct, and NO directly acts on the accessory genital mass which packages eggs. NO also acts at a second site, independent of the effect on the accessory genital mass. A NO donor activates appetitive behaviors that normally precede egg laying even in A. californica that are unable to lay eggs.

A novel ALS-associated variant in UBQLN4 regulates motor axon morphogenesis

The etiological underpinnings of amyotrophic lateral sclerosis (ALS) are complex and incompletely understood, although contributions to pathogenesis by regulators of proteolytic pathways have become increasingly apparent. Here, we present a novel variant in UBQLN4 that is associated with ALS and show that its expression compromises motor axon morphogenesis in mouse motor neurons and in zebrafish. We further demonstrate that the ALS-associated UBQLN4 variant impairs proteasomal function, and identify the Wnt signaling pathway effector beta-catenin as a UBQLN4 substrate. Inhibition of beta-catenin function rescues the UBQLN4 variant-induced motor axon phenotypes. These findings provide a strong link between the regulation of axonal morphogenesis and a new ALS-associated gene variant mediated by protein degradation pathways. DOI: http://dx.doi.org/10.7554/eLife.25453.001

Autaptic muscarinic self-excitation and nitrergic self-inhibition in neurons initiating Aplysia feeding are revealed when the neurons are cultured in isolation

Properties of a neuron may arise via endogenous mechanisms, or via interactions with other neurons. Culturing a neuron in isolation is a useful tool to distinguish between endogenous and circuit-derived properties. We identified two remarkable functional features of pattern initiator neurons B31/B32 in Aplysia when these neurons were cultured in isolation. These features were also present in situ, but were less prominent, and would have been missed had they not been observed first in the isolated cultured neurons. The properties are likely to be present in neurons of higher animals, but have not yet been observed. One feature was autaptic muscarinic self-excitation that contributes to the neuron’s plateau potential, by which it initiates behavior. The other feature was the release of nitric oxide (NO) in the absence of spiking, which causes self-inhibition at rest. The nitrergic modulation of B31/B32 is likely to contribute to the control of feeding by dietary changes in the concentration of l-arginine, the precursor from which NO is synthesized.