Disruption of mitochondrial complex I induces progressive parkinsonism.

Loss of functional mitochondrial complex I (MCI) in the dopaminergic neurons of the substantia nigra is a hallmark of Parkinson's disease1. Yet, whether this change contributes to Parkinson's disease pathogenesis is unclear2. Here we used intersectional genetics to disrupt the function of MCI in mouse dopaminergic neurons. Disruption of MCI induced a Warburg-like shift in metabolism that enabled neuronal survival, but triggered a progressive loss of the dopaminergic phenotype that was first evident in nigrostriatal axons. This axonal deficit was accompanied by motor learning and fine motor deficits, but not by clear levodopa-responsive parkinsonism-which emerged only after the later loss of dopamine release in the substantia nigra. Thus, MCI dysfunction alone is sufficient to cause progressive, human-like parkinsonism in which the loss of nigral dopamine release makes a critical contribution to motor dysfunction, contrary to the current Parkinson's disease paradigm3,4.

Mutant huntingtin enhances activation of dendritic Kv4 K+ channels in striatal spiny projection neurons.

Huntington's disease (HD) is initially characterized by an inability to suppress unwanted movements, a deficit attributable to impaired synaptic activation of striatal indirect pathway spiny projection neurons (iSPNs). To better understand the mechanisms underlying this deficit, striatal neurons in ex vivo brain slices from mouse genetic models of HD were studied using electrophysiological, optical and biochemical approaches. Distal dendrites of iSPNs from symptomatic HD mice were hypoexcitable, a change that was attributable to increased association of dendritic Kv4 potassium channels with auxiliary KChIP subunits. This association was negatively modulated by TrkB receptor signaling. Dendritic excitability of HD iSPNs was rescued by knocking-down expression of Kv4 channels, by disrupting KChIP binding, by restoring TrkB receptor signaling or by lowering mutant-Htt (mHtt) levels with a zinc finger protein. Collectively, these studies demonstrate that mHtt induces reversible alterations in the dendritic excitability of iSPNs that could contribute to the motor symptoms of HD.

Systemic isradipine treatment diminishes calcium-dependent mitochondrial oxidant stress.

The ability of the Cav1 channel inhibitor isradipine to slow the loss of substantia nigra pars compacta (SNc) dopaminergic (DA) neurons and the progression of Parkinson's disease (PD) is being tested in a phase 3 human clinical trial. But it is unclear whether and how chronic isradipine treatment will benefit SNc DA neurons in vivo. To pursue this question, isradipine was given systemically to mice at doses that achieved low nanomolar concentrations in plasma, near those achieved in patients. This treatment diminished cytosolic Ca2+ oscillations in SNc DA neurons without altering autonomous spiking or expression of Ca2+ channels, an effect mimicked by selectively knocking down expression of Cav1.3 channel subunits. Treatment also lowered mitochondrial oxidant stress, reduced a high basal rate of mitophagy, and normalized mitochondrial mass - demonstrating that Cav1 channels drive mitochondrial oxidant stress and turnover in vivo. Thus, chronic isradipine treatment remodeled SNc DA neurons in a way that should not only diminish their vulnerability to mitochondrial challenges, but to autophagic stress as well.

Calcium and Parkinson's disease.

Parkinson's disease (PD) is the second most common neurodegenerative disease in the world. Its causes are poorly understood and there is no proven therapeutic strategy for slowing disease progression. The core motor symptoms of PD are caused by the death of dopaminergic neurons in the substantia nigra pars compacta (SNc). In these neurons, Ca2+entry through plasma membrane Cav1 channels drives a sustained feed-forward stimulation of mitochondrial oxidative phosphorylation. Although this design helps prevent bioenergetic failure when activity needs to be sustained, it leads to basal mitochondrial oxidant stress. Over decades, this basal oxidant stress could compromise mitochondrial function and increase mitophagy, resulting in increased vulnerability to other proteostatic stressors, like elevated alpha synuclein expression. Because this feedforward mechanism is no longer demanded by our lifestyle, it could be dispensed with. Indeed, use of dihydropyridines - negative allosteric modulators of Cav1 Ca2+ channels - comes with little or no effect on brain function but is associated with decreased risk and progression of PD. An ongoing, NIH sponsored, Phase 3 clinical trial in North America is testing the ability of one member of the dihydropyridine class (isradipine) to slow PD progression in early stage patients. The review summarizes the rationale for the trial and outlines some unanswered questions.

Mitochondrial oxidant stress in locus coeruleus is regulated by activity and nitric oxide synthase.

Loss of noradrenergic locus coeruleus (LC) neurons is a prominent feature of aging-related neurodegenerative diseases, such as Parkinson's disease (PD). The basis of this vulnerability is not understood. To explore possible physiological determinants, we studied LC neurons using electrophysiological and optical approaches in ex vivo mouse brain slices. We found that autonomous activity in LC neurons was accompanied by oscillations in dendritic Ca(2+) concentration that were attributable to the opening of L-type Ca(2+) channels. This oscillation elevated mitochondrial oxidant stress and was attenuated by inhibition of nitric oxide synthase. The relationship between activity and stress was malleable, as arousal and carbon dioxide increased the spike rate but differentially affected mitochondrial oxidant stress. Oxidant stress was also increased in an animal model of PD. Thus, our results point to activity-dependent Ca(2+) entry and a resulting mitochondrial oxidant stress as factors contributing to the vulnerability of LC neurons.

Calcium entry induces mitochondrial oxidant stress in vagal neurons at risk in Parkinson's disease.

Mitochondrial oxidant stress is widely viewed as being critical to pathogenesis in Parkinson's disease. But the origins of this stress are poorly defined. One possibility is that it arises from the metabolic demands associated with regenerative activity. To test this hypothesis, we characterized neurons in the dorsal motor nucleus of the vagus (DMV), a population of cholinergic neurons that show signs of pathology in the early stages of Parkinson's disease, in mouse brain slices. DMV neurons were slow, autonomous pacemakers with broad spikes, leading to calcium entry that was weakly buffered. Using a transgenic mouse expressing a redox-sensitive optical probe targeted to the mitochondrial matrix, we found that calcium entry during pacemaking created a basal mitochondrial oxidant stress. Knocking out DJ-1 (also known as PARK7), a gene associated with early-onset Parkinson's disease, exacerbated this stress. These results point to a common mechanism underlying mitochondrial oxidant stress in Parkinson's disease and a therapeutic strategy to ameliorate it.

Oxidant stress evoked by pacemaking in dopaminergic neurons is attenuated by DJ-1.

Parkinson's disease is a pervasive, ageing-related neurodegenerative disease the cardinal motor symptoms of which reflect the loss of a small group of neurons, the dopaminergic neurons in the substantia nigra pars compacta (SNc). Mitochondrial oxidant stress is widely viewed as being responsible for this loss, but why these particular neurons should be stressed is a mystery. Here we show, using transgenic mice that expressed a redox-sensitive variant of green fluorescent protein targeted to the mitochondrial matrix, that the engagement of plasma membrane L-type calcium channels during normal autonomous pacemaking created an oxidant stress that was specific to vulnerable SNc dopaminergic neurons. The oxidant stress engaged defences that induced transient, mild mitochondrial depolarization or uncoupling. The mild uncoupling was not affected by deletion of cyclophilin D, which is a component of the permeability transition pore, but was attenuated by genipin and purine nucleotides, which are antagonists of cloned uncoupling proteins. Knocking out DJ-1 (also known as PARK7 in humans and Park7 in mice), which is a gene associated with an early-onset form of Parkinson's disease, downregulated the expression of two uncoupling proteins (UCP4 (SLC25A27) and UCP5 (SLC25A14)), compromised calcium-induced uncoupling and increased oxidation of matrix proteins specifically in SNc dopaminergic neurons. Because drugs approved for human use can antagonize calcium entry through L-type channels, these results point to a novel neuroprotective strategy for both idiopathic and familial forms of Parkinson's disease.

Recurrent collateral connections of striatal medium spiny neurons are disrupted in models of Parkinson's disease.

The principal neurons of the striatum, GABAergic medium spiny neurons (MSNs), are interconnected by local recurrent axon collateral synapses. Although critical to many striatal models, it is not clear whether these connections are random or whether they preferentially link functionally related groups of MSNs. To address this issue, dual whole patch-clamp recordings were made from striatal MSNs in brain slices taken from transgenic mice in which D(1) or D(2) dopamine receptor expression was reported with EGFP (enhanced green fluorescent protein). These studies revealed that unidirectional connections were common between both D(1) receptor-expressing MSN (D(1) MSN) pairs (26%) and D(2) receptor-expressing MSN (D(2) MSN) pairs (36%). D(2) MSNs also commonly formed synapses on D(1) MSNs (27% of pairs). Conversely, only 6% of the D(1) MSNs formed detectable connections with D(2) MSNs. Furthermore, synaptic connections formed by D(1) MSNs were weaker than those formed by D(2) MSNs, a difference that was attributable to fewer GABA(A) receptors at D(1) MSN synapses. The strength of detectable recurrent connections was dramatically reduced in Parkinson's disease models. The studies demonstrate that recurrent collateral connections between MSNs are not random but rather differentially couple D(1) and D(2) MSNs. Moreover, this recurrent collateral network appears to be disrupted in Parkinson's disease models, potentially contributing to pathological alterations in MSN activity patterns and psychomotor symptoms.

'Rejuvenation' protects neurons in mouse models of Parkinson's disease.

Why dopamine-containing neurons of the brain's substantia nigra pars compacta die in Parkinson's disease has been an enduring mystery. Our studies suggest that the unusual reliance of these neurons on L-type Ca(v)1.3 Ca2+ channels to drive their maintained, rhythmic pacemaking renders them vulnerable to stressors thought to contribute to disease progression. The reliance on these channels increases with age, as juvenile dopamine-containing neurons in the substantia nigra pars compacta use pacemaking mechanisms common to neurons not affected in Parkinson's disease. These mechanisms remain latent in adulthood, and blocking Ca(v)1.3 Ca2+ channels in adult neurons induces a reversion to the juvenile form of pacemaking. Such blocking ('rejuvenation') protects these neurons in both in vitro and in vivo models of Parkinson's disease, pointing to a new strategy that could slow or stop the progression of the disease.

Selective elimination of glutamatergic synapses on striatopallidal neurons in Parkinson disease models.

Parkinson disease is a common neurodegenerative disorder that leads to difficulty in effectively translating thought into action. Although it is known that dopaminergic neurons that innervate the striatum die in Parkinson disease, it is not clear how this loss leads to symptoms. Recent work has implicated striatopallidal medium spiny neurons (MSNs) in this process, but how and precisely why these neurons change is not clear. Using multiphoton imaging, we show that dopamine depletion leads to a rapid and profound loss of spines and glutamatergic synapses on striatopallidal MSNs but not on neighboring striatonigral MSNs. This loss of connectivity is triggered by a new mechanism-dysregulation of intraspine Cav1.3 L-type Ca(2+) channels. The disconnection of striatopallidal neurons from motor command structures is likely to be a key step in the emergence of pathological activity that is responsible for symptoms in Parkinson disease.

Cell death in developing human spinal cord.

Cell death in the developing human spinal cord was investigated in 5-12 week human conceptuses using immunohistochemical and TUNEL methods. Expression of pro-apoptotic (Fas-receptor, caspase-3) and anti-apoptotic (bcl-2) markers and marker for internucleosomal fragmentation (TUNEL) were analysed in the cranial and caudal parts of the human spinal cord. In early developmental stages (5-6 weeks) of the cranial spinal cord, bcl-2 positive cells were seen in the ventricular zone and in the roof plate, while in the caudal part they were seen surrounding the central lumen. Subsequently, bcl-2 expression appeared in the basal plates of the grey matter and in the spinal ganglia, and from the seventh week on they also appeared in the intermediate horn of the grey matter. In the fetal period, bcl-2 expression appeared in the dorsal horns of the grey matter (9 weeks) but ceased in the ventricular zone (12 weeks) . In the trunk region, TUNEL-positive cells were found in ventricular and mantle zones along the whole length of the spinal cord. Caspase-3 positive cells and Fas-receptor positive cells appeared only in the grey matter of the cranial segments (head and trunk) of the spinal cord, but they were missing in the caudal parts. Caspase-3 dependant pathway, probably activated by Fas-receptor, seems to operate only in the cranial part of the human spinal cord. In the caudal (sacrococcygeal and tail) parts, cells seem to die by caspase-3 independent pathway. The interplay of pro-apoptotic and anti-apoptotic factors may be associated with cranial spinal cord morphogenesis, adjustment of cells number and selective survival of neurons, while in the caudal regions these factors cause massive cell death associated with regression of the caudal spinal cord.

Dendritic excitability of mouse frontal cortex pyramidal neurons is shaped by the interaction among HCN, Kir2, and Kleak channels.

Dendritically placed, voltage-sensitive ion channels are key regulators of neuronal synaptic integration. In several cell types, hyperpolarization/cyclic nucleotide gated (HCN) cation channels figure prominently in dendritic mechanisms controlling the temporal summation of excitatory synaptic events. In prefrontal cortex, the sustained activity of pyramidal neurons in working memory tasks is thought to depend on the temporal summation of dendritic excitatory inputs. Yet we know little about how this is accomplished in these neurons and whether HCN channels play a role. To gain a better understanding of this process, layer V-VI pyramidal neurons in slices of mouse prelimbic and infralimbic cortex were studied. Somatic voltage-clamp experiments revealed the presence of rapidly activating and deactivating cationic currents attributable to HCN1/HCN2 channels. These channels were open at the resting membrane potential and had an apparent half-activation voltage near -90 mV. In the same voltage range, K+ currents attributable to Kir2.2/2.3 and K+-selective leak (Kleak) channels were prominent. Computer simulations grounded in the biophysical measurements suggested a dynamic interaction among Kir2, Kleak, and HCN channel currents in shaping membrane potential and the temporal integration of synaptic potentials. This inference was corroborated by experiment. Blockade of Kir2/Kleak channels caused neurons to depolarize, leading to the deactivation of HCN channels, the initiation of regular spiking (4-5 Hz), and enhanced temporal summation of EPSPs. These studies show that HCN channels are key regulators of synaptic integration in prefrontal pyramidal neurons but that their functional contribution is dependent on a partnership with Kir2 and Kleak channels.

Otolith organ or semicircular canal stimulation induces c-fos expression in unipolar brush cells and granule cells of cat and squirrel monkey.

Immediate early gene expression in the cerebellar vermis of cats and squirrel monkeys was stimulated by prolonged whole body rotations. Continuous, earth-horizontal axis rotations that excited only otoliths or high velocity vertical axis rotations that excited only semicircular canals resulted in c-fos immunoreactive nuclei concentrated in the granular layer of lobules X and ventral IX (the nodulus and ventral uvula), which represent the medial parts of the vestibulo-cerebellum. Large clusters of labeled nuclei consisting mainly of granule cells and calretinin-positive unipolar brush cells were present in the granular layer, whereas Purkinje cell nuclei were unlabeled, and labeled basket and stellate cell nuclei were scattered in the molecular layer. In other vermal lobules there was a significant but less dense label than in the nodulus and ventral uvula. Generally, the extent of c-fos labeling of molecular layer interneurons was in relation to nuclear labeling of granular layer neurons: labeling of both basket and stellate cells accompanied nuclear labeling of neurons throughout the depth of the granular layer, whereas only stellate cells were labeled when nuclear labeling was restricted to the superficial granular layer. Yaw horizontal or roll vertical rotations each stimulated c-fos expression in the cat medial vestibulo-cerebellum to approximately the same extent. Low-velocity rotations resulted in much less c-fos expression. Similar, albeit less intense, patterns of c-fos activation were observed in monkeys. Concentrated c-fos expression in the medial vestibulo-cerebellum after exposure to a strong head velocity signal that could originate from either otolith or canal excitation suggests that granule and unipolar brush cells participate in a neuronal network for estimating head velocity, irrespective of the signal source.

G-protein-coupled receptor modulation of striatal CaV1.3 L-type Ca2+ channels is dependent on a Shank-binding domain.

Voltage-gated L-type Ca2+ channels are key determinants of synaptic integration and plasticity, dendritic electrogenesis, and activity-dependent gene expression in neurons. Fulfilling these functions requires appropriate channel gating, perisynaptic targeting, and linkage to intracellular signaling cascades controlled by G-protein-coupled receptors (GPCRs). Surprisingly, little is known about how these requirements are met in neurons. The studies described here shed new light on how this is accomplished. We show that D2 dopaminergic and M1 muscarinic receptors selectively modulate a biophysically distinctive subtype of L-type Ca2+ channels (CaV1.3) in striatal medium spiny neurons. The splice variant of these channels expressed in medium spiny neurons contains cytoplasmic Src homology 3 and PDZ (postsynaptic density-95 (PSD-95)/Discs large/zona occludens-1) domains that bind the synaptic scaffolding protein Shank. Medium spiny neurons coexpressed CaV1.3-interacting Shank isoforms that colocalized with PSD-95 and CaV1.3a channels in puncta resembling spines on which glutamatergic corticostriatal synapses are formed. The modulation of CaV1.3 channels by D2 and M1 receptors was disrupted by intracellular dialysis of a peptide designed to compete for the CaV1.3 PDZ domain but not with one targeting a related PDZ domain. The modulation also was disrupted by application of peptides targeting the Shank interaction with Homer. Upstate transitions in medium spiny neurons driven by activation of glutamatergic receptors were suppressed by genetic deletion of CaV1.3 channels or by activation of D2 dopaminergic receptors. Together, these results suggest that Shank promotes the assembly of a signaling complex at corticostriatal synapses that enables key GPCRs to regulate L-type Ca2+ channels and the integration of glutamatergic synaptic events.

Bromodeoxyuridine administered during neurogenesis of the projection neurons causes cerebellar defects in rat.

Bromodeoxyuridine (BrdU) is broadly used in neuroscience to study embryonic development and adult neurogenesis. The potential toxicity of this halogenated pyrimidine analogue is frequently neglected. In this study, we administered BrdU in small doses by the progressively delayed cumulative labeling method to immunocytochemically tag different cerebellar cell types with antibodies to specific markers and BrdU in the same section. The well-known structure of the cerebellum made it possible to ascertain several toxic effects of the treatment. Time-pregnant rats were given five or six injections of 5 or 6 mg of BrdU ( approximately 12-20 mg/kg) at 8-hour intervals over 2 successive days between day 11 and 21 of pregnancy (E11-E12 to E20-E21), and the adult progeny was processed by immunocytochemistry. We demonstrate that this treatment effectively labeled distinct cerebellar cell populations but produced striking defects in the proliferation, migration, and settling of the Purkinje cells; reduced the size of the cerebellar cortex and nuclei; produced defects in the patterning of foliation; and also affected litter size, body weight, and mortality of the offspring. The observed toxic effects were consistent within individual treatment groups but varied between different treatment groups. Treatment with BrdU at the peak of neurogenesis of cerebellar projection neurons (E14) produced the most severe malformations. We observed no overt effects on the timing of neurogenesis for cerebellar neurons and glia across experimental groups. In conclusion, BrdU is a useful tool to study neural development, but its cytotoxicity represents a serious pitfall particularly when multiple doses are used to label cells.