Neuronal Glial Interactions in Cerebral Energy- and Amino Acid Homeostasis: Implications of Glutamate and GABA

Abstract

Maintenance of cerebral energy metabolism and neurotransmitter amino acid homeostasis are crucial elements for preservation of normal brain function. Hence the scope of this thesis was to elucidate different aspects of these issues while focusing on i) the functional and metabolic role of astrocytic glycogen, ii) the relevance of glucose, lactate and glycogen for fueling glutamatergic neurotransmission and iii) the functional and metabolic roles of the two isoforms of glutamate decarboxylase (GAD), i.e. GAD65 and GAD67.

Glycogen is the only major carbohydrate store in the brain and is confined almost exclusively to astrocytes, providing a readily available energy source upon an increased demand. Degradation of glycogen is activated by an elevation in the intracellular AMP concentration and accordingly is likely to be important for sustaining energy demanding events in the local astrocytic microenvironment. Such a role for glycogen was investigated in cultured astrocytes superfused in the absence or presence of norepinephrine, D-aspartate or an elevated extracellular K+ concentration, i.e. conditions known to increase glycogen metabolism. Metabolism of glucose via glycogen, i.e. glycogen shunt activity, was assessed by employing an inhibitor of glycogen degradation. It was demonstrated that metabolism of glucose by way of the glycogen shunt accounts for a prominent part of astrocytic glucose metabolism. Moreover, it was revealed that glycogen metabolism is enhanced correlating with the level of astrocytic activity, underlining the dynamics of glucose metabolism via the glycogen shunt.

Glutamatergic neurotransmission involves several energy requiring processes in both neurons and astrocytes. Glycogen has repeatedly been demonstrated to be able to sustain neuronal activity in the presence of a physiological glucose concentration and during hypoglycemia and aglycemia. However, whether glycogen is used to fuel processes within astrocytes themselves or is transferred to the neurons in the form of lactate is unclear. Thus, the functional importance of glycogen for energizing glutamate transport was investigated in cultured astrocytes. Moreover, the capacity of glycogen to sustain vesicular release and subsequent synaptic clearance was examined in co-cultures of astrocytes and glutamatergic neurons after repetitive depolarization. It was revealed that glycogen is essential for maintenance of astrocytic transport capacity both in the absence and presence of a physiological glucose concentration. Moreover, glycogen was important for sustaining vesicular release in neurons, at least to some extent by a transfer of lactate from astrocytes to neurons.

Glucose is the primary energy substrate for the mammalian brain; however, lactate produced within the brain, e.g. from glycolysis or glycogen degradation may provide an alternative energy substrate for neurons. Indeed, lactate is extensively metabolized in neurons and is also capable of sustaining neuronal activity. Nonetheless, the capacity of lactate to fuel glutamatergic neurotransmission is debatable. Thus, metabolism of glucose and lactate was investigated in cultured glutamatergic neurons at different levels of activity induced by increasing concentrations of NMDA (0, 30, 100 and 300 μM). It was revealed that glucose consumption and metabolism are correlated with the neuronal activity while lactate metabolism is pronounced but independent of induced depolarization. Moreover, it was demonstrated that NMDA concentration dependently increases the intracellular Ca2+ concentration as well as vesicular release. Based upon the previous findings that a high intracellular Ca2+ concentration inhibits malate-aspartate shuttle activity and increases the cytosolic NADH concentration, a model was proposed to account for the neuronal preference for glucose during depolarization. This model suggests a coupling between the intracellular Ca2+ concentration, the malate-aspartate shuttle activity and the cytosolic NADH level, the latter being determining for the utilization of glucose or lactate as energy substrate. Conversion of both glucose and lactate to pyruvate is associated with NADH production; however, it appears that the conversion of lactate to pyruvate, being dependent only on substrate availability, is more susceptible to an increase in cytosolic NADH than the multiple step pathway of glycolysis.

Finally, the functional impact of GAD65 in maintenance of cerebral GABA homeostasis was examined in vivo using GAD65 knockout and corresponding wild type mice. The two isoforms of GAD, i.e. GAD65 and GAD67, differ with regard to regulation and intracellular distribution, and have therefore been proposed distinct functional roles. It was revealed that GAD65 is important for the synthesis of GABA from astrocytic glutamine specifically by direct conversion of glutamine to GABA via glutamate. In contrast, GAD67 is important for GABA synthesis from glutamine both via direct conversion and by a pathway involving TCA cycle activity. Previous investigations have demonstrated a functional importance of GABA synthesized by GAD65 for sustaining synaptic activity during intense stimulation. Here, the importance of GAD65 for synthesizing GABA destined for tonic inhibition was elucidated in a cortical wedge model and in vivo by administration of kainate, known to augment tonic inhibition. The results demonstrated significant deficits in tonic inhibition in GAD65 knockout mice. Moreover, kainate gave rise to seizure activity in both GAD65 knockout and wild type mice. However, in wild type mice it concomitantly led to severe neuronal hypometabolism, which was not the case in GAD65 knockout mice displaying deficits in tonic inhibition. Based on these findings it was concluded that the hypometabolism induced by kainate in wild type mice results from an increased tonic inhibition mediated by GABA synthesized via GAD65.

Opretholdelse af hjernens energistofskifte og de forskellige puljer af aminosyre neurotransmittere er vigtige elementer for at bevare en normal hjernefunktion. Som folge heraf er formalet med denne afhandling at belyse forskellige aspekter af disse emner, med sarligt fokus pa i) den funktionelle og metaboliske betydning af glykogen i hjernen, ii) relevansen af glukose, glykogen og laktat som neuronale energisubstrater i forbindelse med glutamaterg neurotransmission samt iii) den funktionelle og metaboliske betydning af eksistensen af to isoformer af glutamate decarboxylase (GAD), narmere bestemt GAD65 og GAD67, i hjernen.

Det eneste vasentlige kulhydratlager i hjernen er glykogen, som nasten udelukkende findes i astrocytter. Glykogen udgor et energidepot, der hurtigt kan blive nedbrydes ved et oget energibehov. Nedbrydningen af glykogen aktiveres nar cellens AMP-niveau stiger, og dermed understotter glykogen formentlig energiprocesser i det lokale astrocytiske mikromiljo. Hvorvidt glykogen spiller en sadan funktionel rolle blev undersogt i cellekulturer af astrocytter ved superfusion med medium tilsat noradrenalin, D-aspartat og en hoj K+ koncentration, det vil sige med stoffer med en kendt effekt pa glykogenstofskiftet. Metabolisme af glukose gennem glykogen, dvs. via glykogenshunten, blev vurderet ved samtidig hamning af glykogennedbrydningen. Det blev vist, at glukose metabolisme via glykogenshunten udgor en betragtelig del af den samlede glukosemetabolisme i astrocytter. Derudover var det tydeligt at metabolismen af glykogen i hjernen opreguleres proportionalt med astrocytternes aktivitetsniveau, hvilket understreger at glukosemetabolisme via glykogen er dynamisk.

Glutamaterg neurotransmission bestar af adskillige energikravende processer i bade neuroner og astrocytter. Det er gentagne gange blevet vist at glykogen er i stand til at opretholde neuronal aktivitet bade i tilstedevarelse af en fysiologisk glukose koncentration og under hypoglykami eller aglykami. Derimod er det ikke klarlagt hvorvidt glykogen bliver brugt som energisubstrat for astrocytterne selv eller om det bliver overfort til neuronerne i form af laktat. Det blev derfor undersogt hvorvidt energien fra glykogen bidrager til opretholdelsen af glutamattransporten ind i dyrkede astroctter. Desuden blev det undersogt om glykogen er vigtig for at kunne vedligeholde den vesikulare frisatning fra neuroner og den efterfolgende optagelse af glutamate. Dette blev undersogt i co-kulturer af astrocytter og glutamaterge neuroner ved gentagen depolarisering. Resultatet viste, at glykogen er vigtigt for opretholdelsen af den astrocytiske glutamate transport bade i narvar og fravar af glukose. Derudover blev det klart at glykogen bidrager med energi til bevarelsen af neuronal frisatning, og at dette, i hvert fald i nogen grad, skyldes overforsel af laktat mellem astrocytter og neuroner.

Glukose er det primare energisubstrat i hjernen, men derudover kan laktat produceret via omdannelse af glukose i hjernen vare et alternativt energisubstrat for neuroner. Det er fastslaet at neuroner optager og omdanner vasentlige mangder laktat, og laktat har vist sig at vare i stand til at opretholde neuronal aktivitet. Hvorvidt laktat er i stand til at opretholde glutamaterg neurotransmission er til gengald et omstridt emne. Det blev derfor undersogt hvorledes glukose og laktat metaboliseres i dyrkede glutamaterge neuroner under forskellige grader af aktivitet induceret af stigende koncentrationer af NMDA (0, 30, 100, 300 μM). Optagelsen og metabolismen af glukose viste sig at vare proportional med aktivitetsniveauet mens metabolismen af laktat var uafhangig af depolarisering, pa trods af at en betydelig mangde laktat blev omdannet. Derudover medforte NMDA en koncentrationsafhangig stigning i den intracellulare Ca2+ koncentration samt den vesikulare frisatning. Pa grundlag af tidligere studier, der viste at hoje intracellulare Ca2+ koncentrationer medforer en hamning af malat-aspartat-shuttlen og dermed giver anledning til en foroget cytosolisk NADH koncentration, blev der foreslaet en model, der kan gore rede for at neuronen foretrakker glukose under aktivitet. Denne model foreslar en kobling mellem den intracellulare Ca2+ koncentration, malat-aspartate-shuttle aktivitet samt det cytosoliske NADH niveau, hvor sidstnavnte er bestemmende for hvorvidt glukose eller laktat benyttes som energisubstrat. NADH er et produkt i omdannelsen af bade glukose og laktat til pyruvate, men omdannelsen fra laktat, der udelukkende er afhangig af koncentrationerne af substrater og produkter, synes at blive pavirket mere af en hoj cytosolisk NADH koncentration end glykolysen, der foregar via flere koblede reaktioner.

Til sidst blev det undersogt, ved brug af genmodificerede GAD65 knockout mus, hvilken funktionel betydning GAD65 har for syntesen af GABA og opretholdelsen af GABA puljen i hjernen. De to isoformer af GAD er forskelligt reguleret og lokaliseret intracellulart, og pa grundlag heraf er det foreslaet at de to isoformer har forskellige funktioner. Det blev vist, at GAD65 er vigtig for syntesen af GABA fra astrocytisk glutamin, specifikt ved direkte syntese via glutamat. Derimod er GAD67 vigtig for syntese af GABA fra glutamin bade ved direkte syntese og gennem en syntesevej, der involverer metabolisme i TCA cyklus. Tidligere studier har vist at GABA syntetiseret af GAD65 er vigtig for opretholdelsen af intens synaptisk aktivitet. Her blev det undersogt ved brug af en cortical wedge model, hvorvidt GABA fra GAD65 ogsa er vigtig for den ekstrasynaptiske toniske inhibition. Resultaterne viste, at den toniske hamning er tydeligt formindsket i GAD65 knockout mus. Den metaboliske betydning af dette blev udforsket in vivo ved brug af kainsyre, der tidligere har vist sig at foroge den toniske inhibition. I begge genotyper gav kainsyre anledning til krampeaktivitet. Desuden udviste vildtype mus behandlet med kainsyre en betydelig neuronal hypometabolisme, hvilket ikke var tilfaldet for GAD65 knockout mus, der havde formindsket tonisk inhibition. Pa grundlag af disse resultater blev det konkluderet at hypometabolisme induceret af kainsyre i vildtype mus sker som folge af en oget tonisk inhibition medieret af GABA, der er syntetiseret af GAD65.