Striatal neuronal apoptosis is preferentially enhanced by NMDA receptor activation in YAC transgenic mouse model of Huntington disease

Huntington disease (HD), caused by expansion >35 of a polyglutamine tract in huntingtin, results in degeneration of striatal medium spiny neurons (MSNs). Previous studies demonstrated mitochondrial dysfunction, altered intracellular calcium release, and enhanced NMDAR-mediated current and apoptosis in cellular and mouse models of HD. Here, we exposed cultured MSNs from YAC transgenic mice, expressing full-length human huntingtin with 18, 72, or 128 repeats, to a variety of apoptosis-inducing compounds that inhibit mitochondrial function or increase intracellular calcium, and assessed apoptosis 24 h later. All compounds produced a polyglutamine length-dependent increase in apoptosis, but NMDA produced the largest potentiation in apoptosis of YAC72 and YAC128 versus YAC18 MSNs. Moreover, reduction of NMDAR-mediated current and calcium influx in YAC72 MSNs to levels seen in wild-type reduced NMDAR-mediated apoptosis proportionately to wild-type levels. Our results suggest that increased NMDAR signaling plays a major role in enhanced excitotoxic MSN death in this HD mouse model.     

P38 MAPK is involved in enhanced NMDA receptor-dependent excitotoxicity in YAC transgenic mouse model of Huntington disease

Huntington disease (HD) is a dominantly inherited neurodegenerative disease caused by a polyglutamine (polyQ) expansion in the protein huntingtin (htt). Previous studies have shown enhanced N-methyl-d-aspartate (NMDA)-induced excitotoxicity in neuronal models of HD, mediated in part by increased NMDA receptor (NMDAR) GluN2B subunit binding with the postsynaptic density protein-95 (PSD-95). In cultured hippocampal neurons, the NMDAR-activated p38 Mitogen-activated Protein Kinase (MAPK) death pathway is disrupted by a peptide (Tat-NR2B9c) that uncouples GluN2B from PSD-95, whereas NMDAR-mediated activation of c-Jun N-terminal Kinase (JNK) MAPK is PSD-95-independent. To investigate the mechanism by which Tat-NR2B9c protects striatal medium spiny neurons (MSNs) from mutant htt (mhtt)-enhanced NMDAR toxicity, we compared striatal tissue and cultured MSNs from presymptomatic yeast artificial chromosome (YAC) mice expressing htt with 128 polyQ (YAC128) to those from YAC18 and/or WT mice as controls. Similar to the previously published shift of GluN2B-containing NMDARs to extrasynaptic sites, we found increased PSD-95 localization as well as elevated PSD-95-GluN2B interactions in the striatal non-PSD (extrasynaptic) fraction from YAC128 mice. Notably, basal levels of both activated p38 and JNK MAPKs were elevated in the YAC128 striatum. NMDA stimulation of acute slices increased activation of p38 and JNK in WT and YAC128 striatum, but Tat-NR2B9c pretreatment reduced only the p38 activation in YAC128. In cultured MSNs, p38 MAPK inhibition reduced YAC128 NMDAR-mediated cell death to WT levels, and occluded the Tat-NR2B9c peptide protective effect; in contrast, inhibition of JNK had a similar protective effect in cultured MSNs from both WT and YAC128 mice. Our results suggest that altered activation of p38 MAPK contributes to mhtt enhancement of GluN2B/PSD-95 toxic signaling.     

Full length mutant huntingtin is required for altered Ca2+ signaling and apoptosis of striatal neurons in the YAC mouse model of Huntington's disease

Huntington's disease (HD) is caused by a progressive loss of striatal medium spiny neurons (MSN). The molecular trigger of HD is a polyglutamine expansion in the Huntingtin protein (Htt). The mutant Htt protein forms insoluble nuclear aggregates which have been proposed to play a key role in causing neuronal cell death in HD. Other lines of investigation suggest that expression of mutant Htt facilitates activity of the NR2B subtype of NMDA receptors and the type 1 inositol 1,4,5-trisphosphate receptors (InsP(3)R1), and that disturbed calcium (Ca(2+)) signaling causes apoptosis of MSNs in HD. The YAC128 transgenic HD mouse model expresses the full-length human Htt protein with 120Q CAG repeat expansion and displays an age-dependent loss of striatal neurons as seen in human HD brain. In contrast, the shortstop mice express an amino-terminal fragment of the mutant Htt protein (exons 1 and 2) and display no behavioral abnormalities or striatal neurodegeneration despite widespread formation of neuronal inclusions. Here we compared Ca(2+) signals in primary MSN neuronal cultures derived from YAC128 and shortstop mice to their wild-type non-transgenic littermates. Repetitive application of glutamate results in supranormal Ca(2+) responses in YAC128 MSNs, but not in shortstop MSNs. In addition, while currents mediated by the NR2B subtype of NMDA receptors were increased in YAC128 MSNs, currents in SS MSNs were found to be similar to WT. Furthermore, YAC128 MSNs were sensitized to glutamate-induced apoptosis. Consistent with these findings, we found that application of glutamate induced rapid loss of mitochondrial membrane potential in YAC128 MSNs. In contrast, SS MSNs do not show increased cell death postglutamate treatment nor accelerated loss of mitochondrial membrane potential following glutamate stimulation. Glutamate-induced loss of mitochondrial membrane potential in YAC128 MSNs could be prevented by inhibitors of NR2B NMDA receptors and mGluR1/5 receptors. Our results are consistent with the hypothesis that disturbed neuronal Ca(2+) signaling plays a significant role in the degeneration of MSN containing full-length mutant Htt(exp). Furthermore, the results obtained with neurons from shortstop mice provide additional evidence that not all fragments of mutant Htt(exp) are toxic to neurons.     

Huntington disease: new insights on the role of huntingtin cleavage

Huntington Disease (HD) results from polyglutamine expansion within the N-terminus of huntingtin. We have produced yeast artificial chromosome (YAC) transgenic mice expressing normal (YAC18) and mutant (YAC46 and YAC72) human huntingtin in a developmentally appropriate and tissue-specific manner identical to the pattern of expression of endogenous huntingtin. YAC46 and YAC72 mice show early electrophysiological abnormalities indicating neuronal cytoplasmic dysfunction prior to developing nuclear inclusions or neurodegeneration. YAC72 mice display a hyperkinetic movement disorder by 7 months of age, and have evidence for selective and specific degeneration of medium spiny neurons in the lateral striatum by 12 months of age. A key molecular feature of pathology of these YAC72 mice is cleavage of huntingtin in the cytoplasm following by translocation of the resulting huntingtin N-terminal fragments into the nucleus of striatal neurons. Increasing nuclear localization of huntingtin N-terminal fragments within medium spiny neurons of the striatum occurs concomitantly with the onset of selective neurodegeneration. Because huntingtin is a caspase substrate and truncated huntingtin fragments are toxic in vitro, inhibiting caspase cleavage of huntingtin may be of potential therapeutic benefit in HD. We show that caspase inhibitors eliminate huntingtin cleavage in cells and protects them from an apoptotic stress. We also identify caspase-6 and caspase-3 cleavage sites in huntingtin and demonstrate that neuronal and non-neuronal cells expressing a caspase-resistant huntingtin with an expanded polyglutamine tract are less susceptible to apoptosis and aggregate formation. These results suggest that caspase cleavage of huntingtin may be a crucial step in aggregate formation and neurotoxicity in HD.     

Kainate excitotoxicity in organotypic hippocampal slice cultures: evidence for multiple apoptotic pathways

The mechanisms underlying kainate (KA) neurotoxicity are still not well understood. We previously reported that KA-mediated neuronal damage in organotypic cultures of hippocampal slices was associated with p53 induction. Recently, both bax and caspase-3 have been demonstrated to be key components of the p53-dependent neuronal death pathway. Caspase activation has also been causally related to the release of mitochondrial cytochrome c (Cyto C) in the cytoplasm as a result of the collapse of the mitochondrial membrane potential (Deltapsi(M)) and the opening of mitochondrial permeability transition pores (mPTP). In the present study, we observed a rapid induction of bax in hippocampal slice cultures after KA treatment. In addition, the levels of Cyto C and caspase-3 were increased in the cytosol while the level of the caspase-9 precursor was decreased. There was also a complete reduction of Rhodamine 123 fluorescence after KA treatment, an indication of Deltapsi(M) dissipation. Furthermore, inhibition of mPTP opening by cyclosporin A partially prevented Cyto C release, caspase activation and neuronal death. These data suggest the involvement of bax, several caspases, as well as Cyto C release in KA-elicited neuronal death. Finally, inhibition of caspase-3 activity by z-VAD-fmk only partially protected neurons from KA toxicity, implying that multiple mechanisms may be involved in KA excitotoxicity.     

Age-dependent neurovascular abnormalities and altered microglial morphology in the YAC128 mouse model of Huntington disease

Central nervous system (CNS) inflammatory processes including microglial activation have been implicated in the pathogenesis of neurodegenerative diseases such as Huntington Disease (HD). We report age-dependent changes in striatal microglial morphology and vasculature in the YAC128 mouse model of HD. Decreases in microglial ramification along with a decrease in vessel diameter and increased vessel density and length suggest the presence of microgliosis and proangiogenic activity in YAC128 mice. Our hypothesis for this study was that the changes in microglial morphology and perturbations in vasculature may be involved in the pathogenesis of HD and that peripheral challenge with the bacterial endotoxin, lipopolysaccharide (LPS), will exacerbate these microglial and vascular changes as well as the HD phenotype in YAC128 mice at 12 months. Chronic peripheral LPS (1 mg/kg) potentiated microglial activation indicated by an increase in microglial cell body size and retraction of processes. This potentiation in microglial activation with chronic peripheral LPS challenge was paralleled with vascular remodeling including dilatation, increased vessel wall thickness, increased BBB permeability and fibrinogen deposition in YAC128 striatum. Although peripheral LPS caused an increase in microglial activation and degenerative changes in cerebrovasculature, the phenotypic hallmarks of HD in YAC128 mice such as motor coordination deficits and decreased striatal volume were not exacerbated by chronic peripheral LPS exposure. This study identifies age-dependent increases in microglial activation and angiogenesis in YAC128 at 12 months. Peripheral inflammation induced by chronic LPS causes similar changes but does not influence the HD phenotype in YAC128 mice.     

Cyclosporin A and its nonimmunosuppressive analogue N-Me-Val-4-cyclosporin A mitigate glucose/oxygen deprivation-induced damage to rat cultured hippocampal neurons

When mouse hippocampal neuronal cultures, 2-3 weeks in vitro, were transiently exposed to combined glucose and oxygen deprivation (100% argon, 5% CO2, in glucose-free medium) for 90 min, extensive neuronal degeneration had occurred after 24 h of reoxygenation. When these cultures were preincubated with cyclosporin A, a calcineurin inhibitor and a blocker of the mitochondrial permeability transition, neuronal death diminished by 30-50%. Similarly, the cyclosporin A analogue, N-Me-Val-4-cyclosporin A, a potent blocker of the mitochondrial permeability transition with no significant calcineurin blocking activity, decreased cell death by 70-80%. Both cyclosporin A and N-Me-Val-4-cyclosporin A markedly attenuated calcium-induced swelling of isolated mouse brain mitochondria by blocking the mitochondrial permeability transition. The potassium thiocyanate-stabilized binding of cyclophilin D to mouse brain mitochondrial membranes was completely prevented by cyclosporin A and N-Me-Val-4-cyclosporin A. Our results strongly suggest that the mitochondrial permeability transition is involved in oxygen/glucose deprivation-induced cell death in vitro. Cyclophilin D and other components of the mitochondrial permeability transition may be important targets for neuroprotective and anti-ischaemic drugs.     

Cytochrome C and caspase-9 expression in Huntington's disease

There is increasing evidence implicating apoptosis-mediated cell death in the pathogenesis of neurodegenerative diseases. One important event in the apoptotic cascade is the release of cytochrome c by mitochondria into the cytoplasm, activating caspase-9, leading to the subsequent activation of downstream executioner caspases. In the present study, we examined the distribution of cytochrome c and caspase-9 in Huntington’s disease (HD) patients and in a transgenic model of HD (R6/2 line). Neuronal cytochrome c immunoreactivity increased with neuropathological severity in HD patients. Concomitant with this finding, Western-blot analysis showed a shift in the distribution of cytochrome c from the mitochondrial to the cytosolic fraction with incremental cytosolic expression associated with greater striatal degeneration. Active caspase-9 immunoreactivity was present in both HD striatal neurons and in Western blots of severe-grade specimens. Similar findings were observed in the R6/2 mice. There was a temporal increase in expression and shift of cytochrome c from the mitochondrial to the cytosolic fraction from 4–13 wk of age. Activated caspase-9 and caspase 3 activities were present only at endstage disease. Although the present results provide evidence that key components of the intrinsic mitochondrial apoptotic pathway are activated in both HD patients and a transgene murine model of HD, these phenomena are prominent in only severe neuropathological grades in HD patients and HD mice, suggesting that apoptosis may play a greater role in neuronal death at endstage disease.     

Neuronal gelsolin prevents apoptosis by enhancing actin depolymerization

Gelsolin (gsn), an actin-severing protein, protects neurons from excitotoxic cell death via inactivation of membranous Ca(2+) channels. Its role during apoptotic cell death, however, has remained unclear. Using several models of neuronal cell death, we demonstrate that endogenous gelsolin has anti-apoptotic properties that correlate to its dynamic actions on the cytoskeleton. We show that neurons lacking gelsolin (gsn(-/-)) have enhanced apoptosis following exposure to staurosporine, thapsigargin, or the cholinergic toxin ethylcholine aziridinium (AF64A). AF64A-induced loss of mitochondrial membrane potential and activation of caspase-3 was specifically enhanced in gsn(-/-) neurons and could be reversed by pharmacological inhibition of mitochondrial permeability transition. Moreover, increased caspase-3 activation and cell death in AF64A-treated gsn(-/-) neurons were completely reversed by pharmacological depolymerization of actin filaments and further enhanced by their stabilization. In conclusion, actin remodeling by endogenous gelsolin or analogues protects neurons from apoptosis mediated by mitochondria and caspase-3.     

Mutant huntingtin enhances excitotoxic cell death

Evidence suggests overactivation of NMDA-type glutamate receptors (NMDARs) contributes to selective degeneration of medium-sized spiny striatal neurons in Huntington's disease (HD). Here we determined whether expression of huntingtin containing the polyglutamine expansion augments NMDAR-mediated excitotoxicity. HEK293 cells coexpressing mutant huntingtin (htt-138Q) and either NR1A/NR2A- or NR1A/NR2B-type NMDARs exposed to 1 mM NMDA showed a significant increase in excitotoxic cell death compared to controls (cells coexpressing htt-15Q or GFP), but the difference was larger for NR1A/NR2B. Moreover, agonist-dependent cell death showed apoptotic features for cells coexpressing htt-138Q and NR1A/NR2B, but not for cells expressing htt-138Q and NR1A/NR2A. Further, NR1A/NR2B-mediated apoptosis was not seen with coexpression of an N-terminal fragment of mutant htt. Since NR1A/NR2B is the predominant NMDAR subtype in neostriatal medium-sized spiny neurons, enhancement of NMDA-induced apoptotic death in NR1A/NR2B-expressing cells by full-length mutant htt may contribute to selective neurodegeneration in HD.     

Long-term lentiviral-mediated expression of ciliary neurotrophic factor in the striatum of Huntington's disease transgenic mice

Ciliary neurotrophic factor (CNTF) has been shown to prevent behavioral deficits and striatal degeneration in neurotoxic models of Huntington's disease (HD), but its effect in a genetic model has not been evaluated. Lentiviral vectors expressing the human CNTF or LacZ reporter gene were therefore injected in the striatum of wild-type (WT) and transgenic mice expressing full-length huntingtin with 72 CAG repeats (YAC72). Behavioral analysis showed increased locomotor activity in 5- to 6-month-old YAC72-LacZ mice compared to WT-LacZ animals. Interestingly, CNTF expression reduced the activity levels of YAC72 mice compared to control animals. In both WT and YAC72 mice, CNTF expression was demonstrated in striatal punches, up to a year after lentiviral injection. Stereological analysis revealed that the number of LacZ and DARPP-32-positive neurons were decreased in YAC72-LacZ mice compared to WT-LacZ animals. Assessment of the benefit of CNTF expression in the YAC72 mice was, however, complicated by a down-regulation of DARPP-32 and to a lesser extent of NeuN in all mice treated with CNTF. The expression of the neuronal marker NADPH-d was unaffected by CNTF, but expression of the astrocytic marker glial fibrillary acidic protein (GFAP) was increased. Finally, a reduction of the number of striatal dark cells was observed in YAC mice treated with CNTF compared to LacZ. These data indicate that sustained striatal expression of CNTF can be achieved with lentiviruses. Further studies are, however, needed to investigate the intracellular signaling pathways mediating the long-term effects of CNTF expression on dopamine signaling, glial cell activation and how these changes may affect HD pathology.     

NR2B phosphorylation at tyrosine 1472 in spinal dorsal horn contributed to N-methyl-D-aspartate-induced pain hypersensitivity in mice

Calcium influx via N-methyl-D-aspartate (NMDA)-subtype glutamate receptors (NMDARs) regulates the intracellular trafficking of NMDARs, leading to long-lasting modification of NMDAR-mediated synaptic transmission that is involved in development, learning, and synaptic plasticity. The present study investigated the contribution of such NMDAR-dependent synaptic trafficking in spinal dorsal horn to the induction of pain hypersensitivity. Our data showed that direct activation of NMDARs by intrathecal NMDA application elicited pronounced mechanical allodynia in intact mice, which was concurrent with a specific increase in the abundance of NMDAR subunits NR1 and NR2B at the postsynaptic density (PSD)-enriched fraction. Selective inhibition of NR2B-containing NMDARs (NR2BR) by ifenprodil dose dependently attenuated the mechanical allodynia in NMDA-injected mice, suggesting the importance of NR2BR synaptic accumulation in NMDA-induced pain sensitization. The NR2BR redistribution at synapses after NMDA challenge was associated with a significant increase in NR2B phosphorylation at Tyr1472, a catalytic site by Src family protein tyrosine kinases (SFKs) that has been shown to prevent NR2B endocytosis. Intrathecal injection of a specific SFKs inhibitor, PP2, to block NR2B tyrosine phosphorylation eliminated NMDA-induced NR2BR synaptic expression and also attenuated the mechanical allodynia. These data suggested that activation of spinal NMDARs was able to accumulate NR2BR at synapses via SFK signaling, which might exaggerate NMDAR-dependent nociceptive transmission and contribute to NMDA-induced nociceptive behavioral hyperresponsiveness.     

Blockade of the mitochondrial permeability transition pore diminishes infarct size in the rat after transient middle cerebral artery occlusion.

The mitochondrial permeability transition pore is an inducer of cell death. During the reperfusion phase after cerebral ischemia, calcium accumulates in mitochondria, and a burst of free radical formation occurs, conditions that favor the activation of the mitochondrial permeability transition pore. Here the authors demonstrate that a blocker of the mitochondrial permeability transition pore, the nonimmunosuppressive cyclosporin A analogue N-methyl-Val-4-cyclosporin A (10 mg/kg intraperitoneally), administered during reperfusion and at 24 hours of reperfusion, diminishes infarct size in a rat model of transient focal ischemia of 2 hours' duration. The mitochondrial permeability transition pore may be an important target for drugs against stroke.     

Taurine potentiates presynaptic NMDA receptors in hippocampal Schaffer collateral axons

We have previously shown that activation of presynaptic N-methyl-d-aspartate (NMDA) receptors (NMDAR) enhances the amplitude of the presynaptic fibre volley (FV) evoked in Schaffer collateral axons of rat hippocampal slices, by a mechanism independent of extracellular Ca(2+). Here we compared the pharmacological characteristics of presynaptic NMDARs affecting axon excitability (activated by 10-300 microM NMDA for 10 min), with those mediating field excitatory postsynaptic potentials (NMDA-fEPSP). We found that NMDA-induced potentiation was completely inhibited by NVP-AAM077, an antagonist of NR2A-containing NMDAR, but not by ifenprodil, an NR2B-selective antagonist. The inhibitor of the glycine-binding site in NMDARs, 7-clorokynurenic acid (7-CK), was more potent against NMDA-fEPSP (IC(50) = 6.3 +/- 1.3 microM) than against the NMDA-induced FV potentiation (IC(50) = 26.5 +/- 1.3 microM). Moreover, both post- and presynaptic NMDAR-mediated phenomena were enhanced by glycine and d-serine, but taurine, an endogenous analogue of glycine, only enhanced the latter (EC(50) = 19 microM). Taurine was able to block the inhibitory effect of low doses of 7-CK on NMDA-induced FV potentiation, while glycine and d-serine only reduced the effects of higher concentrations of this drug. Surprisingly, the enhancing effect of taurine on NMDA-induced FV potentiation was blocked when it was co-applied with glycine. Furthermore, the glutamate released synaptically with a train of stimuli also increased FV amplitude by a mechanism dependent on NMDARs; this was potentiated by taurine but not by co-application of taurine and glycine. These results reveal that presynaptic NMDARs have unique properties that mediate the facilitation of axon excitability.     

Intracellular Bax translocation after transient cerebral ischemia: implications for a role of the mitochondrial apoptotic signaling pathway in ischemic neuronal death.

Activation of terminal caspases such as caspase-3 plays an important role in the execution of neuronal cell death after transient cerebral ischemia. Although the precise mechanism by which terminal caspases are activated in ischemic neurons remains elusive, recent studies have postulated that the mitochondrial cell death-signaling pathway may participate in this process. The bcl-2 family member protein Bax is a potent proapoptotic molecule that, on translocation from cytosol to mitochondria, triggers the activation of terminal caspases by increasing mitochondrial membrane permeability and resulting in the release of apoptosis-promoting factors, including cytochrome c. In the present study, the role of intracellular Bax translocation in ischemic brain injury was investigated in a rat model of transient focal ischemia (30 minutes) and reperfusion (1 to 72 hours). Immunochemical studies revealed that transient ischemia induced a rapid translocation of Bax from cytosol to mitochondria in caudate neurons, with a temporal profile and regional distribution coinciding with the mitochondrial release of cytochrome c and caspase-9. Further, in postischemic caudate putamen in vivo and in isolated brain mitochondria in vitro, the authors found enhanced heterodimerization between Bax and the mitochondrial membrane permeabilization-related proteins adenine nucleotide translocator (ANT) and voltage-dependent anion channel. The ANT inhibitor bongkrekic acid prevented Bax and ANT interactions and inhibited Bax-triggered caspase-9 release from isolated brain mitochondria in vitro. Bongkrekic acid also offered significant neuroprotection against ischemia-induced caspase-3 and caspase-9 activation and cell death in the brain. These results strongly suggest that the Bax-mediated mitochondrial apoptotic signaling pathway may play an important role in ischemic neuronal injury.     

Estradiol enhances the neurotoxicity of glutamate in GT1-7 cells through an estrogen receptor-dependent mechanism

Glutamate plays an important role in neuroendocrine regulation of reproduction through acting on the N-methyl-D-asparate receptor (NMDAR) in the preoptic area (POA). However, a larger dose of glutamate is neurotoxic. Estradiol (E2) increases the responsiveness of neurons to glutamate through activation and/or expression of NMDAR. In order to investigate whether estradiol modulates the neurotoxic effect of glutamate on the neurons through estrogen receptor (ER), immortalized GT1-7 cells, which simultaneously express ER and NMDAR were used. Tamoxifen and ICI 182,780, ER antagonist, were used to investigate whether the ER is involved in the effect of estradiol on glutamate-induced neurotoxicity. MK-801, a NMDAR antagonist, was used to confirm the enhancement of NMDAR-mediated neurotoxicity by estradiol. Neurotoxicity was evaluated by cell viability and LDH efflux. Cell death was observed by flow cytometry and DNA fragmentation. The results showed that: (1) estradiol (10 nM, incubated for 3 days) significantly enhanced the glutamate-induced neuronal death; (2) the percentages of necrosis and apoptosis were elevated after glutamate treatment, and estradiol significantly enhanced the glutamate-induced cell death; (3) glutamate-induced DNA fragmentation was enhanced by E2-pretreatment; (4) the induction of cell death and increase of LDH efflux after glutamate treatment were also enhanced by E2-pretreatment; (5) both the tamoxifen and ICI 182,780 abolished the estradiol-enhanced NMDAR expression and neurotoxicity of glutamate; (6) higher dose of MK-801 (2 microM) was needed in E2-pretreated cells than in non-E2-pretreated group to block the glutamate-induced neurotoxicity. These results suggested that pretreatment of estradiol might enhance the expression of NMDAR and subsequent glutamate-induced neurotoxicity on the GT1-7 cells through an ER-dependent manner.     

The Sigma-1 Receptor Mediates Pridopidine Rescue of Mitochondrial Function in Huntington Disease Models

Pridopidine is a selective Sigma-1 receptor (S1R) agonist in clinical development for Huntington disease (HD) and amyotrophic lateral sclerosis. S1R is a chaperone protein localized in mitochondria-associated endoplasmic reticulum (ER) membranes, a signaling platform that regulates Ca²⁺ signaling, reactive oxygen species (ROS) and mitochondrial fission. Here, we investigate the protective effects of pridopidine on various mitochondrial functions in human and mouse HD models. Pridopidine effects on mitochondrial dynamics were assessed in primary neurons from YAC128 HD mice expressing the mutant human HTT gene. We observe that pridopidine prevents the disruption of mitochondria-ER contact sites and improves the co-localization of inositol 1,4,5-trisphosphate receptor (IP₃R) and its chaperone S1R with mitochondria in YAC128 neurons, leading to increased mitochondrial activity, elongation, and motility. Increased mitochondrial respiration is also observed in YAC128 neurons and in pridopidine-treated HD human neural stem cells (hNSCs). ROS levels were assessed after oxidative insult or S1R knockdown in pridopidine-treated YAC128 neurons, HD hNSCs, and human HD lymphoblasts. All HD models show increased ROS levels and deficient antioxidant response, which are efficiently rescued with pridopidine. Importantly, pridopidine treatment before H₂O₂-induced mitochondrial dysfunction and S1R presence are required for HD cytoprotection. YAC128 mice treated at early/pre-symptomatic age with pridopidine show significant improvement in motor coordination, indicating a delay in symptom onset. Additionally, in vivo pridopidine treatment reduces mitochondrial ROS levels by normalizing mitochondrial complex activity. In conclusion, S1R-mediated enhancement of mitochondrial function contributes to the neuroprotective effects of pridopidine, providing insight into its mechanism of action and therapeutic potential.Supplementary InformationThe online version contains supplementary material available at 10.1007/s13311-021-01022-9.     

Inhibition of alpha-ketoglutarate dehydrogenase complex promotes cytochrome c release from mitochondria, caspase-3 activation, and necrotic cell death

Mitochondrial dysfunction has been implicated in cell death in many neurodegenerative diseases. Diminished activity of the alpha-ketoglutarate dehydrogenase complex (KGDHC), a key and arguably rate-limiting enzyme of the Krebs cycle, occurs in these disorders and may underlie decreased brain metabolism. The present studies used alpha-keto-beta-methyl-n-valeric acid (KMV), a structural analogue of alpha-ketoglutarate, to inhibit KGDHC activity to test effects of reduced KGDHC on mitochondrial function and cell death cascades in PC12 cells. KMV decreased in situ KGDHC activity by 52 +/- 7% (1 hr) or 65 +/- 4% (2 hr). Under the same conditions, KMV did not alter the mitochondrial membrane potential (MMP), as assessed with a method that detects changes as small as 5%. KMV also did not alter production of reactive oxygen species (ROS). However, KMV increased lactate dehydrogenase (LDH) release from cells by 100 +/- 4.7%, promoted translocation of mitochondrial cytochrome c to the cytosol, and activated caspase-3. Inhibition of the mitochondrial permeability transition pore (MPTP) by cyclosporin A (CsA) partially blocked this KMV-induced change in cytochrome c (-40%) and LDH (-15%) release, and prevented necrotic cell death. Thus, impairment of this key mitochondrial enzyme in PC12 cells may lead to cytochrome c release and caspase-3 activation by partial opening of the MPTP before the loss of mitochondrial membrane potentials.     

Opposing roles of synaptic and extrasynaptic NMDA receptor signaling in cocultured striatal and cortical neurons

The NMDAR plays a unique and vital role in subcellular signaling. Calcium influx initiates signaling cascades important for both synaptic plasticity and survival; however, overactivation of the receptor leads to toxicity and cell death. This dichotomy is partially explained by the subcellular location of the receptor. NMDARs located at the synapse stimulate cell survival pathways, while extrasynaptic receptors signal for cell death. Thus far, this interplay between synaptic and extrasynaptic NMDARs has been studied exclusively in cortical (CTX) and hippocampal neurons. It was unknown whether other cell types, such as GABAergic medium-sized spiny projection neurons of the striatum (MSNs), which bear the brunt of neurodegeneration in Huntington's disease, follow the same pattern. Here we report synaptic versus extrasynaptic NMDAR signaling in striatal MSNs and resultant activation of cAMP response element binding protein (CREB), in rat primary corticostriatal cocultures. Similarly to CTX, we found in striatal MSNs that synaptic NMDARs activate CREB, whereas extrasynaptic NMDARs dominantly oppose CREB activation. However, MSNs are much less susceptible to NMDA-mediated toxicity than CTX cells and show differences in subcellular GluN2B distribution. Blocking NMDARs with memantine (30 μm) or GluN2B-containing receptors with ifenprodil (3 μm) prevents CREB shutoff effectively in CTX and MSNs, and also rescues both neuronal types from NMDA-mediated toxicity. This work may provide cell and NMDAR subtype-specific targets for treatment of diseases with putative NMDAR involvement, including neurodegenerative disorders and ischemia.