Vascular parkinsonism. / Vaskulær parkinsonisme.

Parkinsonism can have many causes, among them cerebrovascular disease. Vascular parkinsonism can be caused by infarction or haemorrhage in the nigrostriatal pathway, resulting in hemiparkinsonism, or by widespread small vessel disease in the white matter, leading to the gradual development of bilateral symptoms in the lower extremities. Compared to patients with Parkinson's disease, individuals with vascular parkinsonism have earlier onset of gait disturbance, are more likely to have urinary incontinence and cognitive impairment, and have poorer treatment response and prognosis; however, they are less likely to have tremor. With its unclear pathophysiology, varying clinical picture and overlap with other diseases, vascular parkinsonism remains a little known and somewhat controversial diagnosis.

A man in his fifties with increasing motor fluctuations, sleep impairment and altered mental status. / En mann i 50-årene med økende motoriske svingninger, søvnvansker og mental endring.

BACKGROUND/CASE PRESENTATION: A man in his fifties with advanced Parkinson´s disease was admitted with increasing motor fluctuations including dyskinesias, as well as hallucinations and delusions. After reduction of oral dopaminergic treatment, the dyskinesias improved, but the psychotic symptoms persisted. They were perceived as levodopa-induced, despite concurrent prominent bradykinetic-rigid symptoms. Dopaminergic treatment was therefore discontinued. He subsequently developed hyperthermia, severe generalised rigidity and akinesia, and autonomic instability. Parkinsonism-hyperpyrexia syndrome was diagnosed, and continuous intraduodenal levodopa/carbidopa infusion was initiated. Despite this, he had several episodes of respiratory distress requiring mechanical ventilation, as well as bradycardia and a single asystole. Although motor and autonomic dysfunction slowly improved, severe akinetic-rigid and neuropsychiatric symptoms persisted, with poor response to increased levodopa. On vital indication, electroconvulsive therapy was performed with clear improvement of mobility and mental state. A hip fracture requiring surgery necessitated discontinuation of ECT, which failed to show equivalent effect when resumed. His condition was considered terminal and all active treatment ceased, resulting in death a few weeks later. INTERPRETATION: Parkinsonism-hyperpyrexia syndrome can develop if dopaminergic treatment is reduced abruptly and excessively. Coexistence of confusion and/or psychosis and clear bradykinetic-rigid symptoms should alarm the clinician. Dopaminergic treatment should not be discontinued, but given intraduodenally. ECT can be effective if started sufficiently early and administered frequently.

GABA, but Not Bestrophin-1, Is Localized in Astroglial Processes in the Mouse Hippocampus and the Cerebellum.

GABA is proposed to act as a gliotransmitter in the brain. Differences in GABA release from astroglia are thought to underlie differences in tonic inhibition between the cerebellum and the CA1 hippocampus. Here we used quantitative immunogold cytochemistry to localize and compare the levels of GABA in astroglia in these brain regions. We found that the density of GABA immunogold particles was similar in delicate processes of Bergman glia in the cerebellum and astrocytes in the CA1 hippocampus. The astrocytic GABA release is proposed to be mediated by, among others, the Ca2+ activated Cl- channel bestrophin-1. The bestrophin-1 antibodies did not show any significant bestrophin-1 signal in the brain of wt mice, nor in bestrophin-1 knockout mice. The bestrophin-1 signal was low both on Western blots and immunofluorescence laser scanning microscopic images. These results suggest that GABA is localized in astroglia, but in similar concentrations in the cerebellum and CA1 hippocampus, and thus cannot account for differences in tonic inhibition between these brain regions. Furthermore, our data seem to suggest that the GABA release from astroglia previously observed in the hippocampus and cerebellum occurs via mechanisms other than bestrophin-1.

Slc38a1 Conveys Astroglia-Derived Glutamine into GABAergic Interneurons for Neurotransmitter GABA Synthesis.

GABA signaling is involved in a wide range of neuronal functions, such as synchronization of action potential firing, synaptic plasticity and neuronal development. Sustained GABA signaling requires efficient mechanisms for the replenishment of the neurotransmitter pool of GABA. The prevailing theory is that exocytotically released GABA may be transported into perisynaptic astroglia and converted to glutamine, which is then shuttled back to the neurons for resynthesis of GABA-i.e., the glutamate/GABA-glutamine (GGG) cycle. However, an unequivocal demonstration of astroglia-to-nerve terminal transport of glutamine and the contribution of astroglia-derived glutamine to neurotransmitter GABA synthesis is lacking. By genetic inactivation of the amino acid transporter Solute carrier 38 member a1 (Slc38a1)-which is enriched on parvalbumin+ GABAergic neurons-and by intraperitoneal injection of radiolabeled acetate (which is metabolized to glutamine in astroglial cells), we show that Slc38a1 mediates import of astroglia-derived glutamine into GABAergic neurons for synthesis of GABA. In brain slices, we demonstrate the role of Slc38a1 for the uptake of glutamine specifically into GABAergic nerve terminals for the synthesis of GABA depending on demand and glutamine supply. Thus, while leaving room for other pathways, our study demonstrates a key role of Slc38a1 for newly formed GABA, in harmony with the existence of a GGG cycle.

Parkinson's Disease: Can Targeting Inflammation Be an Effective Neuroprotective Strategy?

The reason why dopamine neurons die in Parkinson's disease remains largely unknown. Emerging evidence points to a role for brain inflammation in neurodegeneration. Essential questions are whether brain inflammation happens sufficiently early so that interfering with this process can be expected to slow down neuronal death and whether the contribution from inflammation is large enough so that anti-inflammatory agents can be expected to work. Here I discuss data from human PD studies indicating that brain inflammation is an early event in PD. I also discuss the role of T-lymphocytes and peripheral inflammation for neurodegeneration. I critically discuss the failure of clinical trials targeting inflammation in PD.

Dysfunction of homeostatic control of dopamine by astrocytes in the developing prefrontal cortex leads to cognitive impairments.

Astrocytes orchestrate neural development by powerfully coordinating synapse formation and function and, as such, may be critically involved in the pathogenesis of neurodevelopmental abnormalities and cognitive deficits commonly observed in psychiatric disorders. Here, we report the identification of a subset of cortical astrocytes that are competent for regulating dopamine (DA) homeostasis during postnatal development of the prefrontal cortex (PFC), allowing for optimal DA-mediated maturation of excitatory circuits. Such control of DA homeostasis occurs through the coordinated activity of astroglial vesicular monoamine transporter 2 (VMAT2) together with organic cation transporter 3 and monoamine oxidase type B, two key proteins for DA uptake and metabolism. Conditional deletion of VMAT2 in astrocytes postnatally produces loss of PFC DA homeostasis, leading to defective synaptic transmission and plasticity as well as impaired executive functions. Our findings show a novel role for PFC astrocytes in the DA modulation of cognitive performances with relevance to psychiatric disorders.

Propionate enters GABAergic neurons, inhibits GABA transaminase, causes GABA accumulation and lethargy in a model of propionic acidemia.

Propionic acidemia is the accumulation of propionate in blood due to dysfunction of propionyl-CoA carboxylase. The condition causes lethargy and striatal degeneration with motor impairment in humans. How propionate exerts its toxic effect is unclear. Here, we show that intravenous administration of propionate causes dose-dependent propionate accumulation in the brain and transient lethargy in mice. Propionate, an inhibitor of histone deacetylase, entered GABAergic neurons, as could be seen from increased neuronal histone H4 acetylation in the striatum and neocortex. Propionate caused an increase in GABA (γ-amino butyric acid) levels in the brain, suggesting inhibition of GABA breakdown. In vitro propionate inhibited GABA transaminase with a Ki of ∼1 mmol/l. In isolated nerve endings, propionate caused increased release of GABA to the extracellular fluid. In vivo, propionate reduced cerebral glucose metabolism in both striatum and neocortex. We conclude that propionate-induced inhibition of GABA transaminase causes accumulation of GABA in the brain, leading to increased extracellular GABA concentration, which inhibits neuronal activity and causes lethargy. Propionate-mediated inhibition of neuronal GABA transaminase, an enzyme of the inner mitochondrial membrane, indicates entry of propionate into neuronal mitochondria. However, previous work has shown that neurons are unable to metabolize propionate oxidatively, leading us to conclude that propionyl-CoA synthetase is probably absent from neuronal mitochondria. Propionate-induced inhibition of energy metabolism in GABAergic neurons may render the striatum, in which >90% of the neurons are GABAergic, particularly vulnerable to degeneration in propionic acidemia.

Subcellular expression of aquaporin-4 in substantia nigra of normal and MPTP-treated mice.

Aquaporin-4 (AQP4) is the predominant water channel in mammalian CNS where it is localized at the perivascular astrocytic foot processes abutting brain microvessels. Several lines of evidence suggest that AQP4 is involved in important homeostatic functions and that mislocalization of the perivascular pool of AQP4 is implicated in several different brain disorders. A recent study suggests that the differential susceptibility of midbrain dopaminergic neurons to the parkinsonogenic toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) depends on the expression of AQP4. Further, MRI studies of patients with Parkinson's disease (PD) point to an excessive water accumulation in the substantia nigra (SN). This prompted us to investigate the cellular and subcellular distribution of AQP4 in mouse SN using immunofluorescence and quantitative immunogold cytochemistry. Compared with neocortex, SN exhibits a higher concentration of AQP4. Specifically, judged by electron microscopic immunogold analysis, the perivascular density of AQP4 in SN exceeds by 70% the perivascular density of AQP4 in the neocortex. An even larger difference in AQP4 labeling was found for astrocytic processes in the neuropil. Treatment with MPTP further increased (by >30%) the perivascular AQP4 density in SN, but also increased AQP4 labeling in the neocortex. Our data indicate that the perivascular AQP4 pool in SN is high in normal animals and even higher after treatment with MPTP. This would leave the SN more prone to water accumulation and supports the idea that AQP4 could be involved in the pathogenesis of PD.

Neuroglial Transmission.

Neuroglia, the "glue" that fills the space between neurons in the central nervous system, takes active part in nerve cell signaling. Neuroglial cells, astroglia, oligodendroglia, and microglia, are together about as numerous as neurons in the brain as a whole, and in the cerebral cortex grey matter, but the proportion varies widely among brain regions. Glial volume, however, is less than one-fifth of the tissue volume in grey matter. When stimulated by neurons or other cells, neuroglial cells release gliotransmitters by exocytosis, similar to neurotransmitter release from nerve endings, or by carrier-mediated transport or channel flux through the plasma membrane. Gliotransmitters include the common neurotransmitters glutamate and GABA, the nonstandard amino acid d-serine, the high-energy phosphate ATP, and l-lactate. The latter molecule is a "buffer" between glycolytic and oxidative metabolism as well as a signaling substance recently shown to act on specific lactate receptors in the brain. Complementing neurotransmission at a synapse, neuroglial transmission often implies diffusion of the transmitter over a longer distance and concurs with the concept of volume transmission. Transmission from glia modulates synaptic neurotransmission based on energetic and other local conditions in a volume of tissue surrounding the individual synapse. Neuroglial transmission appears to contribute significantly to brain functions such as memory, as well as to prevalent neuropathologies.

Immunogold characteristics of VGLUT3-positive GABAergic nerve terminals suggest corelease of glutamate.

There is compelling evidence that glutamate can act as a cotransmitter in the mammalian brain. Interestingly, the third vesicular glutamate transporter (VGLUT3) is primarily found in neurons that were anticipated to be nonglutamatergic. Whereas the function of VGLUT3 in acetylcholinergic and serotoninergic neurons has been elucidated, the role of VGLUT3 in neurons releasing gamma-aminobutyric acid (GABA) is not settled. We have previously shown that VGLUT3 is found together with the vesicular GABA transporter (VIAAT) on synaptic vesicle membranes in the hippocampus. Now we provide novel electron microscopic data from the rat hippocampus suggesting that glutamate is enriched in inhibitory nerve terminals containing VGLUT3 compared to those lacking VGLUT3. The opposite was found for GABA; VGLUT3-positive inhibitory terminals contained lower density of GABA labeling compared to VGLUT3-negative inhibitory terminals. In addition, semiquantitative confocal immunofluorescence showed that N-methyl-D-aspartate (NMDA)-receptor labeling was present more frequently in VGLUT3-positive/VIAAT-positive synapses versus in VGLUT3-negative/VIAAT-positive synapses. Electron microscopic immunogold data further suggest that NMDA receptors are enriched in VGLUT3 containing inhibitory terminals. Our data reveal new chemical characteristics of a subset of GABAergic interneurons in the hippocampus. The analyses suggest that glutamate is coreleased with GABA from hippocampal basket cell-synapses to act on NMDA receptors.

Localisation of N-acetylaspartate in oligodendrocytes/myelin.

The role of N-acetylaspartate in the brain is unclear. Here we used specific antibodies against N-acetylaspartate and immunocytochemistry of carbodiimide-fixed adult rodent brain to show that, besides staining of neuronal cell bodies in the grey matter, N-acetylaspartate labelling was present in oligodendrocytes/myelin in white matter tracts. Immunoelectron microscopy of the rat hippocampus showed that N-acetylaspartate was concentrated in the myelin. Also neuronal cell bodies and axons contained significant amounts of N-acetylaspartate, while synaptic elements and astrocytes were low in N-acetylaspartate. Mitochondria in axons and neuronal cell bodies contained higher levels of N-acetylaspartate compared to the cytosol, compatible with synthesis of N-acetylaspartate in mitochondria. In aspartoacylase knockout mice, in which catabolism of N-acetylaspartate is blocked, the levels of N-acetylaspartate were largely increased in oligodendrocytes/myelin. In these mice, the highest myelin concentration of N-acetylaspartate was found in the cerebellum, a region showing overt dysmyelination. In organotypic cortical slice cultures there was no evidence for N-acetylaspartate-induced myelin toxicity, supporting the notion that myelin damage is induced by the lack of N-acetylaspartate for lipid production. Our findings also implicate that N-acetylaspartate signals on magnetic resonance spectroscopy reflect not only vital neurons but also vital oligodendrocytes/myelin.

GABA is localized in dopaminergic synaptic vesicles in the rodent striatum.

Recently, electrophysiological evidence was given for inhibitory postsynaptic responses at dopaminergic striatal synapses. These responses were independent of the vesicular GABA transporter, VGAT, but dependent on the vesicular dopamine transporter VMAT2. The identity and the exact source of the released molecule, as well as the presence of the putative inhibitory transmitter in VMAT2 containing synaptic vesicles remain to be shown. To clarify this, in particular to determine whether GABA is responsible for the inhibitory response at dopaminergic synapses, we used the electron microscopic immunogold method to label in vivo perfusion fixed striatal tissue with antibodies recognising GABA, VGAT, VMAT2 and tyrosine hydroxylase. We show that about 13 % of tyrosine hydroxylase positive and 11 % of VMAT2 axonal terminals in the caudo-putamen contain significant labelling for GABA. Immunogold signals for tyrosine hydroxylase and VGAT was totally segregated into different pools of nerve terminals. Quantitative analyses of the distance between gold particles signalling GABA and synaptic vesicles showed that GABA was as closely associated with synaptic vesicles in tyrosine hydroxylase positive as in tyrosine hydroxylase negative nerve terminals. Likewise, in dopaminergic terminals GABA and VMAT2 immunogold particles showed a close spatial localization, strongly suggesting the presence of GABA in VMAT2 positive synaptic vesicles. Our results suggest that GABA is exocytosed together with dopamine from dopaminergic nerve terminals in the caudo-putamen through VGAT negative and VMAT2 positive synaptic vesicles.

The glia doctrine: addressing the role of glial cells in healthy brain ageing.

Glial cells in their plurality pervade the human brain and impact on brain structure and function. A principal component of the emerging glial doctrine is the hypothesis that astrocytes, the most abundant type of glial cells, trigger major molecular processes leading to brain ageing. Astrocyte biology has been examined using molecular, biochemical and structural methods, as well as 3D brain imaging in live animals and humans. Exosomes are extracelluar membrane vesicles that facilitate communication between glia, and have significant potential for biomarker discovery and drug delivery. Polymorphisms in DNA repair genes may indirectly influence the structure and function of membrane proteins expressed in glial cells and predispose specific cell subgroups to degeneration. Physical exercise may reduce or retard age-related brain deterioration by a mechanism involving neuro-glial processes. It is most likely that additional information about the distribution, structure and function of glial cells will yield novel insight into human brain ageing. Systematic studies of glia and their functions are expected to eventually lead to earlier detection of ageing-related brain dysfunction and to interventions that could delay, reduce or prevent brain dysfunction.

Unaltered lactate and glucose transporter levels in the MPTP mouse model of Parkinson's disease.

BACKGROUND: Metabolic impairment contributes to development of Parkinson's disease (PD). Mitochondrial dysfunction is involved in degeneration of nigral dopamine neurons. Also, in PD there are alterations in glucose metabolism in nigro-striatal pathways, and increased cerebral lactate levels have been found. OBJECTIVES: We raise the question of whether changes in the amount transporters of energy substrates are involved in the pathogenesis of PD. METHODS: We have used confocal immunofluorescence and immunogold postembedding electron microscopic techniques to study whether there are altered levels of the transporters for monocarboxylates (MCT1 and MCT2) and glucose (GLUT1) in the MPTP mouse model of PD. RESULTS: We found that MCT1 and GLUT1 were densely located in blood vessel endothelium, while MCT2 was present in perivascular astrocytic end feet processes in the substantia nigra and the striatum of control mice. We found that the localisation and densities of MCTs and GLUT1 were unaltered in the PD model. DISCUSSION: This is the first study reporting on the distribution of metabolic transporters in PD. Our results suggest that, although there are metabolic impairments in PD, the levels of MCT1, MCT2 and GLUT1 is not changed following dopaminergic neurodegeneration. This is in contrast to findings in other neurodegenerative disease, such as mesial temporal lobe epilepsy, where there are large alterations in MCT levels.

LOC689986, a unique gene showing specific expression in restricted areas of the rodent neocortex.

BACKGROUND: The neocortex is a highly specialised and complex brain structure, involved in numerous tasks, ranging from processing and interpretation of somatosensory information, to control of motor functions. The normal function linked to distinct neocortical areas might involve control of highly specific gene expression, and in order to identify such regionally enriched genes, we previously analysed the global gene expression in three different cortical regions (frontomedial, temporal and occipital cortex) from the adult rat brain. We identified distinct sets of differentially expressed genes. One of these genes, namely the hypothetical protein LOC689986 (LOC689986), was of particular interest, due to an almost exclusive expression in the temporal cortex. RESULTS: Detailed analysis of LOC689986 in the adult rat brain confirmed the expression in confined areas of parieto-temporal cortex, and revealed highly specific expression in layer 4 of the somatosensory cortex, with sharp borders towards the neighbouring motor cortex. In addition, LOC689986 was found to be translated in vivo, and was detected in the somatosensory cortex and in the Purkinje cells of the cerebellar cortex. The protein was present in neuronal dendrites and also in astrocyte cells. Finally, this unique gene is apparently specific for, and highly conserved in, the vertebrate lineage. CONCLUSIONS: In this study, we have partially characterised the highly conserved LOC689986 gene, which is specific to the vertebrate linage. The gene displays a distinct pattern of expression in layer 4 of the somatosensory cortex, and areas of the parieto-temporal cortex in rodents.

Rare contacts between synapses and microglial processes containing high levels of Iba1 and actin--a postembedding immunogold study in the healthy rat brain.

Although microglia is recognised as the cell-mediating innate immunity in the brain, emerging evidence suggests a role of microglia in synaptic communication and modulation. The ability of microglia to move in the neuropil and contact synapses is crucial for such a function. However, the frequency of microglial contact with synapses is not known. Microglia motility is regulated by actin polymerisation and its interaction with ionising calcium-binding adaptor protein 1 (Iba1). In order to move and make contact with synapses, delicate microglial processes should contain high levels of actin and Iba1. To study this we refined an electron microscopic postembedding immunogold method enabling us to identify and quantitatively study different microglial constituents in intact brain tissue. We show that Iba1 and actin were colocalised at high densities in delicate processes in the rat frontal cortex, and that these delicate processes of microglia contact synaptic elements. About 3.5% of the synapses received direct contact from microglia. There was a marked inverse correlation between the densities of Iba1/actin gold particles and the area of the microglial processes, suggesting that the most delicate processes possess the machinery to provide movement in the neuropil. The low frequency of microglia interaction with synaptic elements suggests that microglia have a limited role in overall regulation of synaptic activity.

Vesicular uptake and exocytosis of L-aspartate is independent of sialin.

The mechanism of release and the role of l-aspartate as a central neurotransmitter are controversial. A vesicular release mechanism for l-aspartate has been difficult to prove, as no vesicular l-aspartate transporter was identified until it was found that sialin could transport l-aspartate and l-glutamate when reconstituted into liposomes. We sought to clarify the release mechanism of l-aspartate and the role of sialin in this process by combining l-aspartate uptake studies in isolated synaptic vesicles with immunocyotchemical investigations of hippocampal slices. We found that radiolabeled l-aspartate was taken up into synaptic vesicles. The vesicular l-aspartate uptake, relative to the l-glutamate uptake, was twice as high in the hippocampus as in the whole brain, the striatum, and the entorhinal and frontal cortices and was not inhibited by l-glutamate. We further show that sialin is not essential for exocytosis of l-aspartate, as there was no difference in ATP-dependent l-aspartate uptake in synaptic vesicles from sialin-knockout and wild-type mice. In addition, expression of sialin in PC12 cells did not result in significant vesicle uptake of l-aspartate, and depolarization-induced depletion of l-aspartate from hippocampal nerve terminals was similar in hippocampal slices from sialin-knockout and wild-type mice. Further, there was no evidence for nonvesicular release of l-aspartate via volume-regulated anion channels or plasma membrane excitatory amino acid transporters. This suggests that l-aspartate is exocytotically released from nerve terminals after vesicular accumulation by a transporter other than sialin.

Valproate causes reduction of the excitatory amino acid aspartate in nerve terminals.

Valproate is well established in the treatment of epilepsy and psychiatric disorders, yet the main mechanism of action remains to be determined. Here we show that valproate may reduce neurotransmission of the excitatory amino acid, aspartate. By electron microscopic immunogold cytochemistry we demonstrate a 63-68% reduction in the level of aspartate in excitatory nerve terminals at 30 min after an acute dose of valproate. The level of glutamate in the same terminals was unchanged by valproate treatment. In inhibitory terminals, valproate caused a 65% decrease in the aspartate level, whereas the GABA level was not significantly changed. In summary, the present study shows that valproate reduces the nerve terminal content of the excitatory neurotransmitter aspartate. This points to a new mechanism of action for valproate: reduced neuronal excitation through reduced aspartergic neurotransmission.

A distinct set of synaptic-like microvesicles in atroglial cells contain VGLUT3.

There is increasing evidence for vesicular release of glutamate from astrocytes. We have previously demonstrated existence of VGLUT1 on astrocytic synaptic-like microvesicles (SMLVs) in several brain regions indicating a role in astroglial glutamate release. As VGLUT3 is prominently expressed in non-neuronal cells, this prompted us to investigate whether VGLUT3 is also involved in astroglial release of glutamate. Confocal microscopic investigations revealed that astrocytes in the hippocampus and the frontal cortex, as well as Bergmann glia in the cerebellum were labeled for VGLUT3. Immunogold cytochemistry showed that VGLUT3 gold particles were located over SMLVs in perisynaptic astrocytic and Bergmann glial processes. The specificity of the VGLUT3 immunoreactivity was demonstrated by abolished VGLUT3 labeling in astroglia in VGLUT3 knock-out mice. Double immunogold labeling showed that astrocytic processes contained labeling for VGLUT3 and VGLUT1, but the antibodies labeled separate subpopulations of vesicles in the processes. The ratio of gold particle densities between glial processes and nerve terminals were higher for VGLUT3 than for VGLUT1, suggesting that VGLUT3 is particularly abundant in astrocytic processes. Thus, our data show that VGLUT3 localizes to a distinct set of SMLVs in perisynaptic astroglial processes and suggest that VGLUT3 is important for glutamate release from astrocytes.