The discovery of human of GLUD2 glutamate dehydrogenase and its implications for cell function in health and disease.

While the evolutionary changes that led to traits unique to humans remain unclear, there is increasing evidence that enrichment of the human genome through DNA duplication processes may have contributed to traits such as bipedal locomotion, higher cognitive abilities and language. Among the genes that arose through duplication in primates during the period of increased brain development was GLUD2, which encodes the hGDH2 isoform of glutamate dehydrogenase expressed in neural and other tissues. Glutamate dehydrogenase GDH is an enzyme central to the metabolism of glutamate, the main excitatory neurotransmitter in mammalian brain involved in a multitude of CNS functions, including cognitive processes. In nerve tissue GDH is expressed in astrocytes that wrap excitatory synapses, where it is thought to play a role in the metabolic fate of glutamate removed from the synaptic cleft during excitatory transmission. Expression of GDH rises sharply during postnatal brain development, coinciding with nerve terminal sprouting and synaptogenesis. Compared to the original hGDH1 (encoded by the GLUD1 gene), which is potently inhibited by GTP generated by the Krebs cycle, hGDH2 can function independently of this energy switch. In addition, hGDH2 can operate efficiently in the relatively acidic environment that prevails in astrocytes following glutamate uptake. This adaptation is thought to provide a biological advantage by enabling enhanced enzyme catalysis under intense excitatory neurotransmission. While the novel protein may help astrocytes to handle increased loads of transmitter glutamate, dissociation of hGDH2 from GTP control may render humans vulnerable to deregulation of this enzyme's function. Here we will retrace the cloning and characterization of the novel GLUD2 gene and the potential implications of this discovery in the understanding of mechanisms that permitted the brain and other organs that express hGDH2 to fine-tune their functions in order to meet new challenging demands. In addition, the potential role of gain-of-function of hGDH2 variants in human neurodegenerative processes will be considered.

Allelic Imbalance in TOR1A mRNA Expression in Manifesting and Non-Manifesting Carriers of the GAG-Deletion.

Early onset dystonia (EOD) is associated with a 3bp-(ΔGAG) in-frame deletion in the TOR1A gene, which encodes for torsinA. Carriers of the mutant (ΔGAG) allele can either develop or escape a dystonic phenotype (~30% penetrance). The expression ratio of the two alleles could be important for the manifestation or prevention of the disease since wild-type (WT) torsinA is thought to have protective function. Absence of an antibody discriminating WT from ΔE torsinA has precluded the determination ΔE and WT torsinA levels in manifesting and nonmanifesting carriers. We performed quantitative analysis of TOR1A allele expression in manifesting (MC) and nonmanifesting (NMC) carriers using quantitative allele-specific PCR (qASPCR) to determine the levels of mutant versus WT torsinA mRNA. The technique described showed high degree of specificity in detecting the two alleles. The present study represents the first comprehensive analysis of biallelic expression of the TOR1A gene in lymphoblast and brain samples from patients and NMC relatives. We demonstrate that mRNA is transcribed from both the WT and ΔGAG allele in peripheral and neural tissues with a trend for increased expression of the ΔGAG allele compared to the WT in carriers regardless of their phenotype and thus cannot account for the reduced penetrance.

Reduced pallidal output causes dystonia.

Dystonia is a neurological disorder characterized by sustained or repetitive involuntary muscle contractions and abnormal postures. In the present article, we will introduce our recent electrophysiological studies in hyperkinetic transgenic mice generated as a model of DYT1 dystonia and in a human cervical dystonia patient, and discuss the pathophysiology of dystonia on the basis of these electrophysiological findings. Recording of neuronal activity in the awake state of DYT1 dystonia model mice revealed reduced spontaneous activity with bursts and pauses in both internal (GPi) and external (GPe) segments of the globus pallidus. Electrical stimulation of the primary motor cortex evoked responses composed of excitation and subsequent long-lasting inhibition, the latter of which was never observed in normal mice. In addition, somatotopic arrangements were disorganized in the GPi and GPe of dystonia model mice. In a human cervical dystonia patient, electrical stimulation of the primary motor cortex evoked similar long-lasting inhibition in the GPi and GPe. Thus, reduced GPi output may cause increased thalamic and cortical activity, resulting in the involuntary movements observed in dystonia.

Dysregulation of striatal dopamine release in a mouse model of dystonia.

Dystonia is a neurological disorder characterized by involuntary movements. We examined striatal dopamine (DA) function in hyperactive transgenic (Tg) mice generated as a model of dystonia. Evoked extracellular DA concentration was monitored with carbon-fiber microelectrodes and fast-scan cyclic voltammetry in striatal slices from non-Tg mice, Tg mice with a positive motor phenotype, and phenotype-negative Tg littermates. Peak single-pulse evoked extracellular DA concentration was significantly lower in phenotype-positive mice than in non-Tg or phenotype-negative mice, but indistinguishable between non-Tg and phenotype-negative mice. Phenotype-positive mice also had higher functional D2 DA autoreceptor sensitivity than non-Tg mice, which would be consistent with lower extracellular DA concentration in vivo. Multiple-pulse (phasic) stimulation (five pulses, 10-100 Hz) revealed an enhanced frequency dependence of evoked DA release in phenotype-positive versus non-Tg or phenotype-negative mice, which was exacerbated when extracellular Ca(2+) concentration was lowered. Enhanced sensitivity to phasic stimulation in phenotype-positive mice was reminiscent of the pattern seen with antagonism of nicotinic acetylcholine receptors. Consistent with a role for altered cholinergic regulation, the difference in phasic responsiveness among groups was lost when nicotinic receptors were blocked by mecamylamine. Together, these data implicate compromised DA release regulation, possibly from cholinergic dysfunction, in the motor symptoms of this dystonia model.

Cortically evoked long-lasting inhibition of pallidal neurons in a transgenic mouse model of dystonia.

Dystonia is a neurological disorder characterized by sustained or repetitive involuntary muscle contractions and abnormal postures. To understand the pathophysiology of dystonia, neurophysiological analyses were performed on hyperkinetic transgenic mice generated as a model of DYT1 dystonia. Abnormal muscle activity, such as coactivation of agonist and antagonist muscles and sustained muscle activation, was frequently observed in these mice. Recording of neuronal activity in the awake state revealed reduced spontaneous activity with bursts and pauses in both the external and internal segments of the globus pallidus. Motor cortical stimulation evoked responses composed of excitation and subsequent long-lasting inhibition in both pallidal segments, which were never observed in the normal mice. In addition, the somatotopic arrangements in both pallidal segments were disorganized. Long-lasting inhibition induced by cortical inputs in the internal pallidal segment may disinhibit thalamic and cortical activity, resulting in the motor hyperactivity observed in the transgenic mice.

Regulation of DYT1 gene expression by the Ets family of transcription factors.

The DYT1 gene encodes for torsinA, a protein with widespread tissue distribution, involved in early onset dystonia (EOD). Numerous studies have focused on torsinA function but no information is available on its transcriptional regulation. We cloned mouse and human 5'-upstream DYT1 DNA fragments, exhibiting high transcriptional activity, as well as tissue specificity. We identified a proximal minimal DYT1 promoter within -141 bp for mouse and -191 bp for human with respect to the ATG codon. Primer extension analysis indicated multiple transcription start sites. In silico analysis of approximately 500 bp 5'-upstream DYT1 fragment demonstrated lack of a classical TATA or CAAT box and the presence of a highly conserved direct repeat of two Ets binding cores within -86 bp to -77 bp and -78 bp to -69 bp of the mouse and human DYT1 gene, respectively. A single or a two base nucleotide alteration within the downstream Ets core resulted in approximately 90% (mouse) or 45-60% (human) drop in activity. Interestingly, a 3-bp distance increase between the two Ets cores dramatically decreased transcriptional activity which was partially restored when the distance was increased up to 10 bp. Ets-like dominant negatives confirmed the Ets factors as DYT1 transcriptional activators.

Brainstem pathology in DYT1 primary torsion dystonia.

DYT1 dystonia is a severe form of young-onset dystonia caused by a mutation in the gene that encodes for the protein torsinA, which is thought to play a role in protein transport and degradation. We describe, for the first time to our knowledge, perinuclear inclusion bodies in the midbrain reticular formation and periaqueductal gray in four clinically documented and genetically confirmed DYT1 patients but not in controls. The inclusions were located within cholinergic and other neurons in the pedunculopontine nucleus, cuneiform nucleus, and griseum centrale mesencephali and stained positively for ubiquitin, torsinA, and the nuclear envelope protein lamin A/C. No evidence of inclusion body formation was detected in the substantia nigra pars compacta, striatum, hippocampus, or selected regions of the cerebral cortex. We also noted tau/ubiquitin-immunoreactive aggregates in pigmented neurons of the substantia nigra pars compacta and locus coeruleus in all four DYT1 dystonia cases, but not in controls. This study supports the notion that DYT1 dystonia is associated with impaired protein handling and the nuclear envelope. The role of the pedunculopontine and cuneiform nuclei, and related brainstem brainstem structures, in mediating motor activity and controlling muscle tone suggests that alterations in these structures could underlie the pathophysiology of DYT1 dystonia [corrected]