The clinical landscape of cell-free DNA alterations in 1671 patients with advanced biliary tract cancer.

BACKGROUND: Targeted therapies have transformed clinical management of advanced biliary tract cancer (BTC). Cell-free DNA (cfDNA) analysis is an attractive approach for cancer genomic profiling that overcomes many limitations of traditional tissue-based analysis. We examined cfDNA as a tool to inform clinical management of patients with advanced BTC and generate novel insights into BTC tumor biology. PATIENTS AND METHODS: We analyzed next-generation sequencing data of 2068 cfDNA samples from 1671 patients with advanced BTC generated with Guardant360. We carried out clinical annotation on a multi-institutional subset (n = 225) to assess intra-patient cfDNA-tumor concordance and the association of cfDNA variant allele fraction (VAF) with clinical outcomes. RESULTS: Genetic alterations were detected in cfDNA in 84% of patients, with targetable alterations detected in 44% of patients. Fibroblast growth factor receptor 2 (FGFR2) fusions, isocitrate dehydrogenase 1 (IDH1) mutations, and BRAF V600E were clonal in the majority of cases, affirming these targetable alterations as early driver events in BTC. Concordance between cfDNA and tissue for mutation detection was high for IDH1 mutations (87%) and BRAF V600E (100%), and low for FGFR2 fusions (18%). cfDNA analysis uncovered novel putative mechanisms of resistance to targeted therapies, including mutation of the cysteine residue (FGFR2 C492F) to which covalent FGFR inhibitors bind. High pre-treatment cfDNA VAF was associated with poor prognosis and shorter response to chemotherapy and targeted therapy. Finally, we report the frequency of promising targets in advanced BTC currently under investigation in other advanced solid tumors, including KRAS G12C (1.0%), KRAS G12D (5.1%), PIK3CA mutations (6.8%), and ERBB2 amplifications (4.9%). CONCLUSIONS: These findings from the largest and most comprehensive study to date of cfDNA from patients with advanced BTC highlight the utility of cfDNA analysis in current management of this disease. Characterization of oncogenic drivers and mechanisms of therapeutic resistance in this study will inform drug development efforts to reduce mortality for patients with BTC.

Protection of ischemic rat spinal cord white matter: Dual action of KB-R7943 on Na+/Ca2+ exchange and L-type Ca2+ channels.

The effect of the Na+/Ca(2+)-exchange inhibitor KB-R7943 was investigated in spinal cord dorsal column ischemia in vitro. Oxygen/glucose deprivation at 37 degrees C for 1 h causes severe injury even in the absence of external Ca2+. KB-R7943 was very protective in the presence and absence of external Ca2+ implicating mechanisms in addition to extracellular Ca2+ influx through Na+/Ca(2+)-exchange, such as activation of ryanodine receptors by L-type Ca2+ channels. Indeed, blockade of L-type Ca2+ by nimodipine confers a certain degree of protection of dorsal column against ischemia; combined application of nimodipine and KB-R7943 was not additive suggesting that KB-R7943 may also act on Ca2+ channels. KB-R7943 reduced inward Ba2+ current with IC50 = 7 microM in tsA-201 cells expressing Ca(v)1.2. Moreover, nifedipine and KB-R7943 both reduced depolarization-induced [Ca2+]i increases in forebrain neurons and effects were not additive. Nimodipine or KB-R7943 also reduced ischemic axoplasmic Ca2+ increase, which persisted in 0Ca2+/EGTA perfusate in dorsal column during ischemia. While KB-R7943 cannot be considered to be a specific Na+/Ca2+ exchange inhibitor, its profile makes it a very useful neuroprotectant in dorsal columns by: reducing Ca2+ import through reverse Na+/Ca2+ exchange; reducing influx through L-type Ca2+ channels, and indirectly inhibiting Ca2+ release from the ER through activation of ryanodine receptors.

Repolarization of the plasma membrane shapes NMDA-induced cytosolic [Ca2+ ] transients.

After inactivation of NMDA receptors, restoration of basal cytosolic [Ca2+] ([Ca2+]c) is delayed. This may be caused by Ca2+ influx via reverse Na/Ca exchange or voltage-gated Ca2+ channels, and/or by Ca2+ efflux from internal stores. Monitoring of [Na+]c, [Ca2+]c, and plasma membrane potential in cultured cerebellar granule cells showed that repolarization of the plasma membrane and inactivation of voltage-gated Ca channels plays the most critical role in restoration of low [Ca2+]c following NMDA receptor inactivation. During NMDA receptor activation, however, an Na-dependent mechanism enhanced NMDA-induced elevation in [Ca2+]c. This mechanism did not involve Na,K-ATPase activation by Na+, because it operated even when Na,K-ATPase was inhibited.

Kainate-induced K+ efflux and plasma membrane depolarization in cultured cerebellar granule cells.

It is becoming increasingly evident that activation of ionotropic glutamate receptors leads to significant changes in cytosolic [K+] ([K+]c), a major determinant of the plasma membrane (PM) potential, Em. Since Em affects fluxes of key cations, such as Ca2+, it is important to precisely quantify [K+]c and Em in neurons exposed to glutamate receptor agonists. Here we studied the relationships between [K+]c and Em in primary cultures of cerebellar granule cells, and found that kainate elicits a rapid drop in [K+]c below 10 mM. Using patch electrodes containing 10 or 150 mM K+, we determined that kainate depolarizes the PM to -2 or -28 mV, respectively. Therefore, the actual PM depolarization elicited by kainate is much larger than that routinely measured with K+-rich electrodes.

Elevated extracellular K(+) concentrations inhibit N-methyl-D-aspartate-induced Ca(2+) influx and excitotoxicity.

Although extracellular [K(+)] ([K(+)](E)) is highly elevated during brain ischemia, in vitro studies aimed at explaining the mechanisms of excitotoxicity have been conducted at low [K(+)](E). Whether high [K(+)](E) affects excitotoxicity has not been formally addressed. Therefore this study, using digital fluorescence microscopy, tested how the elevation of [K(+)](E) from 5.6 to 60 mM affects N-methyl-D-aspartate (NMDA)-induced Ca(2+) and Na(+) influx, plasma membrane (PM) potential, mitochondrial Ca(2+) load, and viability of primary cultures of rat cerebellar granule cells. High [K(+)](E) curtailed the NMDA-induced Ca(2+) and Na(+) influx and mitochondrial Ca(2+) overload, and prevented neuronal death. Surprisingly, the inhibitory effect of high [K(+)](E) on the NMDA-induced Ca(2+) influx could not be linked to depolarization of the PM. Apparently, the PM of cerebellar granule cells exposed to NMDA was more depolarized at low than at high [K(+)](E), probably because the NMDA-induced Na(+) influx was greatly enhanced when the extracellular [Na(+)]/[K(+)] ratio was increased. When this ratio was small, i.e., at high [K(+)](E), the NMDA-induced increase in cytoplasmic [Na(+)] was suppressed, preventing Ca(2+) influx via the reverse operation of the Na(+)/Ca(2+) exchanger, which may explain the inhibitory effect of high [K(+)](E) on NMDA-induced Ca(2+) influx and excitotoxicity.

N-methyl-D-aspartate excitotoxicity: relationships among plasma membrane potential, Na(+)/Ca(2+) exchange, mitochondrial Ca(2+) overload, and cytoplasmic concentrations of Ca(2+), H(+), and K(+).

A high cytoplasmic Na(+) concentration may contribute to N-methyl-D-aspartate (NMDA)-induced excitotoxicity by promoting Ca(2+) influx via reverse operation of the Na(+)/Ca(2+) exchanger (NaCaX), but may simultaneously decrease the electrochemical Ca(2+) driving force by depolarizing the plasma membrane (PM). Digital fluorescence microscopy was used to compare the effects of Na(+) versus ions that do not support the NaCaX operation, i.e., N-methyl-D-glucamine(+) or Li(+), on: PM potential; cytoplasmic concentrations of Ca(2+), H(+), and K(+); mitochondrial Ca(2+) storage; and viability of primary cultures of cerebellar granule cells exposed to NMDA receptor agonists. In the presence of Na(+) or Li(+), NMDA depolarized the PM and decreased cytoplasmic pH (pH(C)); in the presence of Li(+), Ca(2+) influx was reduced, mitochondrial Ca(2+) overload did not occur, and the cytoplasm became more acidified than in the presence of Na(+). In the presence of N-methyl-D-glucamine(+), NMDA instantly hyperpolarized the PM, but further changes in PM potential and pH(C) were Ca-dependent. In the absence of Ca(2+), hyperpolarization persisted, pH(C) was decreasing very slowly, K(+) was retained in the cytoplasm, and cerebellar granule cells survived the challenge; in the presence of Ca(2+), pH(C) dropped rapidly, the K(+) concentration gradient across the PM began to collapse as the PM began to depolarize, and Ca(2+) influx and excitotoxicity greatly increased. These results indicate that the dominant, very likely excitotoxic, component of NMDA-induced Ca(2+) influx is mediated by reverse NaCaX and that direct Ca(2+) influx via NMDA channels is curtailed by Na-dependent PM depolarization.

The difference between mechanisms of kainate and glutamate excitotoxicity in vitro: osmotic lesion versus mitochondrial depolarization.

The hypothesis that a destabilization of mitochondrial function during neuronal exposure to excitatory amino acids may be involved in the mechanism of neuronal death was examined. The mitochondrial membrane potential (Delta(psi)m) and the cytoplasmic Ca2+ concentration ([Ca2+]c) were monitored simultaneously in single cultured rat cerebellar granule cells (CGCs) loaded with tetramethylrhodamine methyl ester (TMR) and fura-2; CGCs were depolarized with K+, or exposed to excitotoxic doses of glutamate or kainate, and viability of the same neurons was studied for 24-30 h. This approach made it possible to single out the neurons that died, and to describe the changes in Delta(psi)m and [Ca2+]c that were characteristic for these neurons. Exposure to glutamate caused an increase in [Ca2+]c that was associated with a decrease in the mitochondrial TMR fluorescence, which indicates a decrease in Delta(psi)m. The neurons that failed to restore Delta(psi)m following glutamate withdrawal, also failed to restore low [Ca2+]c, and later died. Although a similar number of neurons died following kainate exposure as did after glutamate exposure, the kainate-elicited neuronal death resulted not from the collapse of Delta(psi)m but from an excessive neuronal swelling, which led to rupture of the plasma membrane. Depolarization with K+ was not neurotoxic and caused only a minor decrease in TMR fluorescence. These results indicate that in vitro glutamate and kainate destroy neurons by different mechanisms: glutamate by a failure to restore Delta(psi)m following the exposure, and kainate by an osmotic lesion of the plasma membrane.

Glutamate-induced destabilization of intracellular calcium concentration homeostasis in cultured cerebellar granule cells: role of mitochondria in calcium buffering.

The exposure of cultured cerebellar granule cells for 4 min to glutamate (50 microM) in a Mg2+-free medium containing 10 microM glycine elicited a prompt increase of the intracellular Ca2+ concentration ([Ca2+]i) to 5 microM, which was followed by a decline to 1.5 microM (as measured using fura-2); both events occurred while the glutamate pulse increased the intracellular sodium concentration ([Na+]i) to an estimated 60-100 mM. Because under these circumstances the plasma membrane Na+/Ca2+ exchanger cannot extrude Ca2+, other mechanisms should operate in causing the [Ca2+]i decline. To evaluate a possible role of intracellular Ca2+ stores in Ca2+ buffering, thapsigargin, ryanodine, and dantrolene were tested. Thapsigargin (1 microM) and ryanodine (10 microM) failed to modify the glutamate-elicited [Ca2+]i transients; results with dantrolene could not be considered because this drug by itself affected the fura-2 fluorescence. In contrast, carbonyl cyanide m-chlorophenylhydrazone (1 microM) and antimycin A1 (1 microM), which dissipate mitochondrial membrane potential by different mechanisms, virtually abolished the [Ca2+]i decline occurring either during glutamate application or after its removal. Moreover, when the residual [Na+]i increase persisting after glutamate removal was artificially abated, the Ca2+-buffering capacity of neurons was significantly improved. These data suggest that most of the Ca2+ entering the neurons during excitotoxic glutamate exposure is diverted to mitochondria and that the glutamate-induced increase of [Na+]i limits this mitochondrial Ca2+-buffering capacity, presumably via activation of the mitochondrial Na+/Ca2+ exchanger.

Intracellular sodium concentration in cultured cerebellar granule cells challenged with glutamate.

We monitored simultaneously the changes in the intracellular sodium concentration ([Na+]i) and intracellular calcium concentration ([Ca2+]i) in individual neurons from primary cultures of cerebellar granule cells loaded with sodium-binding benzofuran isophthalate and fluo-3. An application of glutamate (50 microM) in Mg(2+)-free medium containing 10 microM glycine evoked [Na+]i and [Ca2+]i increases that exceeded 60 mM and 1 microM, respectively. The kinetics of [Na+]i and [Ca2+]i decreases after the termination of the glutamate pulse were different. [Na+]i failed to decrease immediately after glutamate withdrawal and the delay in the onset of [Na+]i decrease after the glutamate pulse termination was proportional to the glutamate dose, the glutamate pulse duration, and the extent of [Ca2+]i elevation elicited by glutamate. The kinetics of [Ca2+]i decrease were biphasic, with the first phase occurring immediately after glutamate withdrawal and the second phase being correlated in time with a [Na+]i value lower than 15-20 mM. These results were interpreted to indicate that the glutamate-evoked calcium influx may lead to sodium homeostasis destabilization. The delay in the restoration of the sodium gradient may in turn prolong the neuronal exposure to toxic [Ca2+]i values, due to the decrease in the efficiency of the Na+/Ca2+ exchanger to extrude calcium. The glutamate effects on [Na+]i and [Ca2+]i were potentiated by glycine. Glycine (10 microM) added alone also evoked [Na+]i and [Ca2+]i increases; this effect was inhibited by a competitive inhibitor of the N-methyl-D-aspartate receptor, 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid, indicating an involvement of endogenous glutamate.

Glutamate impairs neuronal calcium extrusion while reducing sodium gradient.

The rate of decrease of neuronal [Ca2+]i after an elevation induced by a glutamate pulse is much slower than that after a comparable [Ca2+]i elevation induced by a K+ depolarization. To investigate whether the [Na+]i increase taking place during the glutamate pulse reduces the rate of Ca2+ extrusion, we monitored simultaneously [Na+]i and [Ca2+]i during a K+ depolarization and a glutamate pulse lasting 1 min. The K+ depolarization evoked only a transient increase of [Na+]i from 4 mM to 13 mM, whereas the glutamate pulse increased [Na+]i to 60 mM, and this increase persisted after glutamate removal. An application of bepridil immediately after glutamate pulse when [Na+]i was greatly elevated, but not 14 min after glutamate removal when a basal [Na+]i was restored, evoked a [Ca2+]i increase accompanied by a decrease of [Na+]i, indicating a reverse mode of operation of the Na+/Ca2+ exchanger. These data suggest that the glutamate-evoked increase in [Na+]i may play a role in Ca2+ homeostasis destabilization.

Sodium nitroprusside inhibits N-methyl-D-aspartate-evoked calcium influx via a nitric oxide- and cGMP-independent mechanism.

In primary cultures of rat cerebellar granule cells, sodium nitroprusside (SNP), a vasodilator that generates nitric oxide (NO), potently inhibited N-methyl-D-aspartate (NMDA)-evoked 45Ca2+ influx (IC50 = 6.6 microM). This inhibition was time dependent and was complete when SNP was applied 10 min before NMDA stimulation. The effect of SNP was transient and the ability of NMDA to stimulate 45Ca2+ influx was restored after SNP withdrawal. The effect of SNP was selective for the NMDA-sensitive glutamate receptor, because SNP failed to antagonize kainate-stimulated 45Ca2+ influx. The action of SNP was independent of the ability of this agent to generate NO; S-nitroso-N-acetylpenicillamine, an NO-containing compound that was 100 times more potent than SNP in stimulating cGMP accumulation, failed to inhibit NMDA-evoked 45Ca2+ influx. In contrast, K4Fe(CN)6, a compound structurally similar to SNP but devoid of NO, inhibited both 45Ca2+ influx (IC50 = 27 microM) and cGMP accumulation evoked by NMDA; K3Fe(CN)6 was inactive. Thus, in cerebellar granule cells, SNP and K4Fe(CN)6 interfere with the function of NMDA receptors, possibly at the level of the receptor recognition site. The resulting blockade of Ca2+ influx through NMDA receptor channels accounts for the reported ability of these compounds to protect granule cells from NMDA-induced neurotoxicity. This protection is not mediated by an NO-dependent mechanism but depends on the action of the ferrocyanide portion of the SNP molecule.

Glutamate receptor agonists stimulate nitric oxide synthase in primary cultures of cerebellar granule cells.

The glutamate receptor agonist N-methyl-D-aspartate (NMDA) stimulated a rapid, extracellular Ca(2+)-dependent conversion of [3H]arginine to [3H]citrulline in primary cultures of cerebellar granule cells, indicating receptor-mediated activation of nitric oxide (NO) synthase. The NMDA-induced formation of [3H]citrulline reached a plateau within 10 min. Subsequent addition of unlabeled L-arginine resulted in the disappearance of 3H from the citrulline pool, indicating a persistent activation of NO synthase after NMDA receptor stimulation. Glutamate, NMDA, and kainate, but not quisqualate, stimulated both the conversion of [3H]arginine to [3H]citrulline and cyclic GMP accumulation in a dose-dependent manner. Glutamate and NMDA showed similar potencies for the stimulation of [3H]citrulline formation and cyclic GMP synthesis, respectively, whereas kainate was more potent at inducing cyclic GMP accumulation than at stimulating [3H]citrulline formation. Both the [3H]arginine to [3H]citrulline conversion and cyclic GMP synthesis stimulated by NMDA were inhibited by the NMDA receptor antagonist MK-801 and by the inhibitors of NO synthase, NG-monomethyl-L-arginine (MeArg) and NG-nitro-L-arginine (NOArg). However, MeArg, in contrast to NOArg, also potently inhibited [3H]arginine uptake. Kainate (300 microM) stimulated 45Ca2+ influx to the same extent as 100 microM NMDA, but stimulated [3H]citrulline formation to a much lesser extent, which suggests that NO synthase is localized in subcellular compartments where the Ca2+ concentration is regulated mainly by the NMDA receptor.

In vitro interaction between cerebellar astrocytes and granule cells: a putative role for nitric oxide.

In primary cultures of astrocytes and granule cells from neonatal rat cerebellum, the activity and function of nitric oxide (NO) synthase were measured by the conversion of [3H]arginine to [3H]citrulline and the accumulation of cyclic guanosine monophosphate (cGMP), respectively. The glutamate receptor agonist N-methyl-D-aspartate (NMDA) and the Ca2+ ionophore A23187 stimulated NO synthase activity in cerebellar granule cells but not in astrocytes. In granule cells, NMDA, A23187, and sodium nitroprusside (SNP) elicited an accumulation of cGMP, whereas only SNP was active in astrocytes. However, in astrocytes that were incubated together with granule cells, NMDA induced a more than 3-fold increase in the concentration of cGMP; this increase was blocked by both the NO synthase inhibitor NG-monomethyl-L-arginine (MeArg) and the allosteric NMDA receptor antagonist (+)5-methyl-10,11-dihydro-5H-dibenzocyclohepten-5,10-imine maleate (MK-801). Thus, cerebellar astrocytes do not appear to express NO synthase but do contain guanylate cyclase, which can be activated by an NO-like factor produced by cerebellar granule cells after stimulation by NMDA.

Inhibition of glutamate-induced cell death by sodium nitroprusside is not mediated by nitric oxide.

Pretreatment of primary cultures of cerebellar granule cells with sodium nitroprusside (SNP) protected these neurons from delayed death induced by glutamate and N-methyl-D-aspartate (NMDA). This neuroprotective effect was not mimicked by S-nitroso-N-acetylpenicillamine (SNAP) which like SNP stimulates guanylate cyclase via a nitric oxide (NO) related mechanism. In contrast, neuroprotection was achieved with potassium ferrocyanide, a compound structurally related to SNP, but devoid of NO. On the other hand, kainate-induced neurotoxicity was not protected but potentiated by SNP. This effect of SNP was not mimicked by SNAP, potassium ferrocyanide and potassium ferricyanide. We conclude that neuroprotective properties of SNP on glutamate- and NMDA-induced neurotoxicity are not due to the release of NO and activation of guanylate cyclase, but are determined by the ferrocyanide portion of the SNP molecule.

The distribution of serotonergic 5-HT1A and non-5-HT1A recognition sites along the dorso-ventral axis of the rat hippocampus.

The distribution of serotonergic 5-HT1 binding sites along the hippocampal dorso-ventral axis was compared with [3H]5-HT uptake considered as a marker of the quantity of serotonergic nerve terminals. [3H]5-HT binding to 5-HT1A and non-5-HT1A sites, of high and low affinity for spiperone, respectively, was investigated in three parts of the hippocampus: dorsal, medial and ventral. In each hippocampal part, 5-HT1A sites constituted approximately 70% of binding sites. [3H]5-HT uptake and [3H]5-HT binding to 5-HT1A and non-5-HT1A sites were significantly higher in the ventral than in the dorsal part of the hippocampus. The dorso-ventral hippocampal gradient in [3H]5-HT binding to both 5-HT1A and non-5-HT1A sites was due to an increase of the density (Bmax) of these sites along the longitudinal hippocampal axis, but not their affinity (Kd) for [3H]5-HT. The results are discussed in the context of the previously described mismatch between the localization of serotonergic nerve terminals and serotonergic receptors in the hippocampus.

Differential response of subtypes of 5-HT1 recognition sites in the rat hippocampus to partial denervation.

[3H]5-HT uptake and [3H]5-HT binding to 5-HT1 receptor subtypes (5-HT1A and non-5-HT1A sites, having high and low affinity to spiperone, respectively) were studied in the rat hippocampus ten days after two types of electrocoagulative lesions. The lesion of supracallosal area and subtotal lesion of the septum destroy supra- and subcallosal hippocampal afferents, respectively. Both types of afferents carry serotonergic fibers to the hippocampus. A significant decrease of [3H]5-HT uptake (by about one half of the control) was observed after both lesions. [3H]5-HT binding to 5-HT1A sites, comprising ca 60% of the total number of 5-HT1 sites, remained unchanged after both lesions, while the binding to non-5-HT1A sites decreased significantly (by ca 20%) but only after the lesion of the septum. The results point to a postsynaptic localization of 5-HT1A and of the bulk of non-5-HT1A sites; the decrease of the proportion of non-5-HT1A sites after septal lesion may be due to their presynaptic localization on the subcallosal pathway and/or may reflect receptor alterations in consequence of transsynaptic events in the hippocampus caused by septal lesion. Differential response of serotonin receptor subtypes to lesions of supracallosal and septal areas may underlie the differential functional responses to those lesions.

Time dependent effect of GM, ganglioside administration upon [(3)H]5-hydroxytryptamine uptake in partially denervated rat hippocampus.

The effect of GM(1) administration upon the time course of the postlesion changes of [(3)H]5hydroxytryptamine uptake in the partially denervated rat hippocampus has been studied 6, 21, 42 and 90 days after surgery. At 6 and 21 days after operation the postlesion decrease of [(3)H]5-hydroxytryptamine uptake was counteracted by GM(1) administration. This effect was limited to the most denervated dorsal hippocampal part, where the lesion-evoked decrease of [(3)H]5-hydroxytryptamine uptake was the strongest. After longer postoperative periods (42 and 90 days) the potentiating effect of GM(1) disappeared. On the other hand, prolonged GM(1) administration (21-90 days) to unoperated animals caused a small but significant reduction of [(3)H]5-hydroxytryptamine uptake, equal in all three hippocampal parts. The early postlesion facilitation of [(3)H]5-hydroxytryptamine uptake due to GM(1) treatment was most probably related to protection against secondary degeneration of serotonergic fibers not directly affected by the lesion.

The effect of GM1 ganglioside on cholinergic and serotoninergic systems in the rat hippocampus following partial denervation is dependent on the degree of fiber degeneration.

The partial lesion paradigm of the dorsal hippocampal afferents in the rat was used as a model to study the effect of GM1 ganglioside treatment on recovery of neurotransmitter markers of the cholinergic and serotoninergic activity in various hippocampal regions. It was found that the enhancement of recovery of acetylcholinesterase, choline acetyltransferase and serotonin uptake by GM1 treatment (30 mg/kg i.m., daily), as studied on the 6th and 21st postlesion day, was dependent on the degree of fiber degeneration. The results may be interpreted in terms of the relationship between the action of GM1 and that of neuronotrophic factors whose release also depends on the extent of the fiber degeneration. These data indicate that GM1 elicits the recovery of biochemical parameters, or fails to, depending on the specificity of the trauma. The result may explain why, after certain brain lesions, GM1 does not promote functional recovery.