P. Shashidharan

Impairment of the ubiquitin-proteasome system causes dopaminergic cell death and inclusion body formation in ventral mesencephalic cultures

Mutations in α‐synuclein, parkin and ubiquitin C‐terminal hydrolase L1, and defects in 26/20S proteasomes, cause or are associated with the development of familial and sporadic Parkinsons disease (PD). This suggests that failure of the ubiquitin–proteasome system (UPS) to degrade abnormal proteins may underlie nigral degeneration and Lewy body formation that occur in PD. To explore this concept, we studied the effects of lactacystin‐mediated inhibition of 26/20S proteasomal function and ubiquitin aldehyde (UbA)‐induced impairment of ubiquitin C‐terminal hydrolase (UCH) activity in fetal rat ventral mesencephalic cultures. We demonstrate that both lactacystin and UbA caused concentration‐dependent and preferential degeneration of dopaminergic neurons. Inhibition of 26/20S proteasomal function was accompanied by the accumulation of α‐synuclein and ubiquitin, and the formation of inclusions that were immunoreactive for these proteins, in the cytoplasm of VM neurons. Inhibition of UCH was associated with a loss of ubiquitin immunoreactivity in the cytoplasm of VM neurons, but there was a marked and localized increase in α‐synuclein staining which may represent the formation of inclusions bodies in VM neurons. These findings provide direct evidence that impaired protein clearance can induce dopaminergic cell death and the formation of proteinaceous inclusion bodies in VM neurons. This study supports the concept that defects in the UPS may underlie nigral pathology in familial and sporadic forms of PD.

Aggresome‐related biogenesis of Lewy bodies

Neurodegenerative disorders such as Parkinsons disease (PD) and ‘dementia with Lewy bodies’ (DLB) are characterized pathologically by selective neuronal death and the appearance of intracytoplasmic protein aggregates (Lewy bodies). The process by which these inclusions are formed and their role in the neurodegenerative process remain elusive. In this study, we demonstrate a close relationship between Lewy bodies and aggresomes, which are cytoplasmic inclusions formed at the centrosome as a cytoprotective response to sequester and degrade excess levels of potentially toxic abnormal proteins within cells. We show that the centrosome/aggresome‐related proteins γ‐tubulin and pericentrin display an aggresome‐like distribution in Lewy bodies in PD and DLB. Lewy bodies also sequester the ubiquitin‐activating enzyme (E1), the proteasome activators PA700 and PA28, and HSP70, all of which are recruited to aggresomes for enhanced proteolysis. Using novel antibodies that are specific and highly sensitive to ubiquitin–protein conjugates, we revealed the presence of numerous discrete ubiquitinated protein aggregates in neuronal soma and processes in PD and DLB. These aggregates appear to be being transported from peripheral sites to the centrosome where they are sequestered to form Lewy bodies in neurons. Finally, we have shown that inhibition of proteasomal function or generation of misfolded proteins cause the formation of aggresome/Lewy body‐like inclusions and cytotoxicity in dopaminergic neurons in culture. These observations suggest that Lewy body formation may be an aggresome‐related event in response to increasing levels of abnormal proteins in neurons. This phenomenon is consistent with growing evidence that altered protein handling underlies the etiopathogenesis of PD and related disorders.

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 other involved brainstem structures in mediating motor activity and muscle tone also suggest that alterations in these structures, in mediating motor activity and controlling muscle tone suggests that alterations in these structures could underlie the pathophysiology of DYT1 dystonia. Ann Neurol 2004

Glutamate transport and metabolism in dopaminergic neurons of substantia nigra: implications for the pathogenesis of Parkinson’s disease

Abstract Parkinson’s disease (PD) is associated with degeneration of the pigmented dopaminergic neurons located in the ventral mesencephalon. Although the mechanisms by which these neurons degenerate in PD are poorly understood, indirect evidence suggests involvement of glutamatergic mechanisms in the pathogenesis of this disorder. Glutamate, the major excitatory transmitter in the mammalian central nervous system, is known to be neurotoxic when present in excess at the synapses. Two major mechanisms protect neurons from glutamate-induced toxicity: (a) removal of synaptic glutamate via a high affinity uptake carried out by cytoplasmic membrane proteins known as excitatory amino acid transporters (EAAT); and (b) metabolism and recycling of glutamate by synaptic astrocytes via glutamine synthetase, an ATP-requiring reaction. However, when extra-cellular glutamate levels are high (0.5–1.0 mM), glutamate metabolism may be shifted toward the ATP-generating oxidative deamination (glutamate dehydrogenase)-TCA cycle pathway. We have cloned and characterized two human glutamate dehydrogenases (GDH), one of which is nerve tissue specific. This isoenzyme requires ADP for its activity and it may become functional when cellular energy charge is low. We have also cloned three human glutamate transporters. One of these (EAAT3) is neuron specific. In situ hybridization studies using human brain revealed that the pigmented dopaminergic neurons, which degenerate in PD, express EAAT3 at high levels. Primary nerve tissue cultures derived from rat ventral mesencephalon were established and studied for their ability to metabolize glutamate. Results showed that mature cultures expressing high levels of GDH activity were capable of rapidly utilizing glutamate added to the medium at high concentrations (1–1.2 mM). This was associated with little release of aspartate and alanine into the medium. In contrast, immature cultures expressing low GDH activity utilized glutamate at lower rates while releasing substantial amounts of aspartate and alanine into the medium. These data suggest that immature mesencephalic cells metabolize a substantial fraction of the glutamate they take up from the medium via the transamination pathway, compared to mature mesencephalic cultures. Immunocytochemical studies on these cultures revealed that dopaminergic neurons (identified by their tyrosine hydroxylase content) showed intense staining for GDH. Furthermore, inhibition of GDH expression by antisense oligonucleotides was toxic to cultured mesencephalic neurons, with dopaminergic neurons being affected at the early stages of this inhibition. Hence, the dense expression by dopaminergic neurons of proteins in-volved in the transport and metabolism of glutamate may serve particular biological needs intrinsic to these cells. Further studies are required to test whether these properties render these neurons vulnerable to excitotoxic mechanisms or to abnormalities of glutamate metabolism.

TorsinA accumulation in Lewy bodies in sporadic Parkinson's disease.

Parkinsons disease (PD) is a neurodegnerative disorder that is pathologically characterized by the presence of Lewy bodies in the brain. We show that Lewy bodies in PD are strongly immunoreactive for torsinA, the protein product of the DYT1 gene, which is associated with primary generalized dystonia. In the substantia nigra, torsinA immunoreactivity is localized to the periphery of Lewy bodies, whereas, in cortical Lewy bodies it is uniformly distributed. The significance of this finding is unknown, but may implicate torsinA in neuronal dysfunction that occurs in PD as well as in primary dystonia.

Immunohistochemical localization and distribution of torsinA in normal human and rat brain

Dystonia is a disease of basal ganglia function, the pathophysiology of which is poorly understood. Primary torsion dystonia is one of the most severe types of inherited dystonia and can be transmitted in an autosomal dominant manner. Recently, one mutation causing this disorder was localized to a gene on chromosome 9q34, designated DYT1, which encodes for a novel protein termed torsinA. The role of this protein in cellular function, in either normal or dystonic individuals is not known. We have developed a polyclonal antibody to torsinA and report its localization and distribution in normal human and rat brain. We demonstrate that torsinA is widely expressed in brain and peripheral tissues. Immunohistochemical studies of normal human and rat brain reveal the presence of torsinA in the dopaminergic neurons of the substantia nigra pars compacta (SNc), in addition to many other regions, including neocortex, hippocampus, and cerebellum. Labeling is restricted to neurons, as shown by double-immunofluorescence microscopy, and is present in both nuclei and cytoplasm. An ATP-binding property for torsinA has been suggested by its homology to ATP-binding proteins; this was confirmed by enrichment of torsinA in ATP-agarose affinity-purified fractions from tissue homogenates. An understanding of the role of torsinA in cellular function and the impact of the mutation (deletion of a glutamic acid at residue 303) is likely to provide insights into the etiopathogenesis of primary dystonia.

Immunohistochemical localization of the neuron-specific glutamate transporter EAAC1 (EAAT3) in rat brain and spinal cord revealed by a novel monoclonal antibody

Neuronal regulation of glutamate homeostasis is mediated by high-affinity sodium-dependent and highly hydrophobic plasma membrane glycoproteins which maintain low levels of glutamate at central synapses. To further elucidate the molecular mechanisms that regulate glutamate metabolism and glutamate flux at central synapses, a monoclonal antibody was produced to a synthetic peptide corresponding to amino acid residues 161-177 of the deduced sequence of the human neuron-specific glutamate transporter III (EAAC1). Immunoblot analysis of human and rat brain total homogenates and isolated synaptosomes from frontal cortex revealed that the antibody immunoreacted with a protein band of apparent Mr approximately 70 kDa. Deglycosylation of immunoprecipitates obtained using the monoclonal antibody yielded a protein with a lower apparent Mr (approximately 65 kDa). These results are consistent with the molecular size of the human EAAC1 predicted from the cloned cDNA. Analysis of the transfected COS-1 cells by immunocytochemistry confirmed that the monoclonal antibody is specific for the neuron-specific glutamate transporter. Immunocytochemical studies of rat cerebral cortex, hippocampus, cerebellum, substantia nigra and spinal cord revealed intense labeling of neuronal somata, dendrites, fine-caliber fibers and puncta. Double-label immunofluorescence using antibody to glial fibrillary acidic protein as a marker for astrocytes demonstrated that astrocytes were not co-labeled for EAAC1. The localization of EAAC1 immunoreactivity in dendrites and particularly in cell somata suggests that this transporter may function in the regulation of other aspects of glutamate metabolism in addition to terminating the action of synaptically released glutamate at central synapses.

Nerve tissue-specific (GLUD2) and housekeeping (GLUD1) human glutamate dehydrogenases are regulated by distinct allosteric mechanisms: implications for biologic function.

Abstract: Human glutamate dehydrogenase (GDH), an enzyme central to the metabolism of glutamate, is known to exist in housekeeping and nerve tissue‐specific isoforms encoded by the GLUD1 and GLUD2 genes, respectively. As there is evidence that GDH function in vivo is regulated, and that regulatory mutations of human GDH are associated with metabolic abnormalities, we sought here to characterize further the functional properties of the two human isoenzymes. Each was obtained in recombinant form by expressing the corresponding cDNAs in Sf9 cells and studied with respect to its regulation by endogenous allosteric effectors, such as purine nucleotides and branched chain amino acids. Results showed that L‐leucine, at 1.0 mM, enhanced the activity of the nerve tissue‐specific (GLUD2‐derived) enzyme by ∼1,600% and that of the GLUD1‐derived GDH by ∼75%. Concentrations of L‐leucine similar to those present in human tissues (∼0.1 mM) had little effect on either isoenzyme. However, the presence of ADP (10‐50 μM) sensitized the two isoenzymes to L‐leucine, permitting substantial enzyme activation at physiologically relevant concentrations of this amino acid. Nonactivated GLUD1 GDH was markedly inhibited by GTP (IC50 = 0.20 μM), whereas nonactivated GLUD2 GDH was totally insensitive to this compound (IC50 > 5,000 μM). In contrast, GLUD2 GDH activated by ADP and/or L‐leucine was amenable to this inhibition, although at substantially higher GTP concentrations than the GLUD1 enzyme. ADP and L‐leucine, acting synergistically, modified the cooperativity curves of the two isoenzymes. Kinetic studies revealed significant differences in the Km values obtained for α‐ketoglutarate and glutamate for the GLUD1‐ and the GLUD2‐derived GDH, with the allosteric activators differentially altering these values. Hence, the activity of the two human GDH is regulated by distinct allosteric mechanisms, and these findings may have implications for the biologic functions of these isoenzymes.

Molecular cloning of human brain glutamate/aspartate transporter II

Glutamate transporters are membrane-bound proteins which are localized in glial cells and/or pre-synaptic glutamatergic nerve endings and are essential for the removal and termination of action of synaptic glutamate. Several cDNAs encoding glutamate transporters have been isolated from mammalian tissues, including human cerebellum. Here, we screened cDNA libraries derived from human brain stem and cerebellum, and isolated a novel cDNA that encodes for a glutamate transporter. This cDNA predicts a protein which contains 565 amino acids and is homologous to a rat brain Na(+)-dependent glutamate/aspartate transporter. The new cDNA is expressed in brain and is structurally distinct from the previously reported human glutamate transporter cDNA.

Nerve tissue-specific human Glutamate dehydrogenase that is thermolabile and highly regulated by ADP

Abstract: Glutamate dehydrogenase (GDH), an enzyme that is central to the metabolism of glutamate, is present at high levels in the mammalian brain. Studies on human leukocytes and rat brain suggested the presence of two GDH activities differing in thermal stability and allosteric regulation, but molecular biological investigations led to the cloning of two human GDH‐specific genes encoding highly homologous polypeptides. The first gene, designated GLUD1, is expressed in all tissues (housekeeping GDH), whereas the second gene, designated GLUD2, is expressed specifically in neural and testicular tissues. In this study, we obtained both GDH isoenzymes in pure form by expressing a GLUD1 cDNA and a GLUD2 cDNA in Sf9 cells and studied their properties. The enzymes generated showed comparable catalytic properties when fully activated by 1 mM ADP. However, in the absence of ADP, the nerve tissue‐specific GDH showed only 5% of its maximal activity, compared with ∼40% showed by the housekeeping enzyme. Low physiological levels of ADP (0.05–0.25 mM) induced a concentration‐dependent enhancement of enzyme activity that was proportionally greater for the nerve tissue GDH (by 550–1,300%) than of the housekeeping enzyme (by 120–150%). Magnesium chloride (1–2 mM) inhibited the nonactivated housekeeping GDH (by 45–64%); this inhibition was reversed almost completely by ADP. In contrast, Mg2+ did not affect the nonstimulated nerve tissue‐specific GDH, although the cation prevented much of the allosteric activation of the enzyme at low ADP levels (0.05–0.25 mM). Heat‐inactivation experiments revealed that the half‐life of the housekeeping and nerve tissue‐specific GDH was 3.5 and 0.5 h, respectively. Hence, the nerve tissue‐specific GDH is relatively thermolabile and has evolved into a highly regulated enzyme. These allosteric properties may be of importance for regulating brain glutamate fluxes in vivo under changing energy demands.