Tenuigenin promotes non-rapid eye movement sleep via the GABAA receptor and exerts somnogenic effect in a MPTP mouse model of Parkinson's disease.

Sleep disturbances are commonly non-motor symptoms in Parkinson's diseases (PD). However, standard dopamine replacement therapies for the treatment of motor symptoms often prove inadequate in combating sleep disturbances. Previous studies conducted by our research group have reported the neuroprotective effects of tenuigenin, a natural extract from Polygala tenuifolia root, which has been traditionally employed in treating insomnia. The objective of this study was to investigate the potential of tenuigenin in modulating sleep-wake behaviors and elucidate the underlying mechanisms. We employed EEG/EMG recordings to evaluate the impact of tenuigenin on sleep-wake profiles. Furthermore, we utilized c-Fos immunostaining, whole-cell patch clamping and local field potentials (LFP) recording to explore the mechanisms involved in sleep-promoting effects of tenuigenin. Additionally, we examined the effects of tenuigenin on sleep-promoting in MPTP PD mice. Here, we found tenuigenin demonstrated a significant increase in NREM sleep and a reduction in sleep latency in mice, without altering the EEG power density. Moreover, tenuigenin increased c-Fos expression in the ventrolateral preoptic area (VLPO) and stimulated sleep-promoting neurons in VLPO. The sleep-promoting effects of tenuigenin were abolished when mice were pretreated with flumazenil, an antagonist at the benzodiazepine site of the GABAA receptor. Furthermore, tenuigenin was found to ameliorate sleep disturbances in MPTP-induced mice. The results suggesting that tenuigenin facilitated a type of NREM sleep comparable to physiological NREM sleep through interaction with the GABAA receptor. Additionally, tenuigenin demonstrated improvements in sleep disturbances in MPTP-induced PD mice, suggesting its potential as a sleep-promoting substance, particularly for PD patients experiencing sleep disturbances.

Seizure Characteristics and Background Amplitude-Integrated Electroencephalography Activity in Neonatal Rats Subjected to Hypoxia-Ischemia.

Objective: Perinatal hypoxic-ischemic encephalopathy (HIE) is a major cause of epilepsy and chronic neurologic morbidity in premature infants. This study aimed to investigate the characteristics of acute seizures and the pattern of background activity on amplitude-integrated electroencephalography (aEEG) in neonatal rats with HIE. Methods: Hypoxia-ischemia (HI) was induced in postnatal day (P) 3 neonatal rats (n = 12) by ligation of the left carotid artery and exposure to airtight hypoxia for 2 h. Data regarding seizure type, frequency, and duration and those related to neurobehavioral development were collected, and the integrated power of background EEG was analyzed to evaluate the effect of HI. Results: All neonatal rats in the HI group experienced frequent seizures during hypoxia, and 83.3% of rats (10/12) experienced seizures immediately after hypoxia. Seizure frequency and duration gradually decreased with increasing age. The mortality rate of the HI group was 8.33% (1/12); 120 h after HI induction, only 27.3% (3/11) of pups had low-frequency and short-duration electrographic seizures, respectively. HI rats, which presented seizure activities 96 h after HI insult, exhibited an increase in righting reflex time and a decrease in forelimb grip reflex time. Background EEG was significantly inhibited during HI induction and immediately after hypoxia and gradually recovered 72 h after hypoxia. Conclusion: Seizures caused by HI brain damage in premature infants can be simulated in the P3 neonatal rat model.

Longitudinal Analysis of Sleep-Wake States in Neonatal Rats Subjected to Hypoxia-Ischemia.

Objective: Sleep is necessary for brain maturation in infants. Perinatal hypoxic-ischemic encephalopathy (HIE) is a major cause of chronic neurological disease in infants. Although the developmental changes of electroencephalogram (EEG) in human newborns have been described, little is known about the EEG normal maturation characteristics in rodents and the changes in sleep-awake states caused by hypoxia-ischemia (HI). This study aimed to investigate the pathological response of sleep-wake states in neonatal rats with HIE. Methods: We constructed HIE and sham models on postnatal day (P) 3 rats and continuously monitored them using electroencephalography and electromyography for up to P12. The distribution of sleep-wake states was analyzed to estimate the effects of HIE. Results: Compared with the sham group, the HI group showed lower rapid eye movement (REM) sleep percentage, but wake percentage and frequency was higher during P4-P12. The frequency of REM and non-rapid eye movement (NREM) sleep increased and the duration of REM and NREM sleep decreased after HI induction. However, it gradually returned to the normal level with an increase in daytime. Conclusion: HI damage alters the sleep-wake patterns during early neural development. The findings provide a comprehensive assessment of serial sleep-wake state recordings in neonatal rats from P4-P12.

Specific Knockdown of α-Synuclein by Peptide-Directed Proteasome Degradation Rescued Its Associated Neurotoxicity.

α-Synuclein (α-syn) overload is strongly associated with Parkinson disease (PD), and reduction of the α-syn level by targeting the peptide-based system through the autophagy-lysosomal pathway (ALP) is a promising strategy to delay PD progression. However, if the ALP is comprised, targeting the peptide-based proteasomal degradation system would be a good alternative. In this study, we designed a fusion peptide containing an α-syn-binding domain and a short strong proteasome-targeting motif. Our results reveal that this peptide could specifically bind to α-syn, and direct it to the proteasomes for degradation in a recombinant expression system. Furthermore, by adding a membrane-penetrating motif to this fusion peptide, we demonstrated that it could penetrate into cells and consequently suppress the cellular α-syn level through proteasome degradation in a dose- and time-dependent manner. Functionally, these effects rescued the mitochondrial dysfunction and cellular defects caused by α-syn overexpression in the cultured cells and primary neurons.

Exosomal DNA Aptamer Targeting α-Synuclein Aggregates Reduced Neuropathological Deficits in a Mouse Parkinson's Disease Model.

The α-synuclein aggregates are the main component of Lewy bodies in Parkinson's disease (PD) brain, and they showed immunotherapy could be employed to alleviate α-synuclein aggregate pathology in PD. Recently we have generated DNA aptamers that specifically recognize α-synuclein. In this study, we further investigated the in vivo effect of these aptamers on the neuropathological deficits associated with PD. For efficient delivery of the aptamers into the mouse brain, we employed modified exosomes with the neuron-specific rabies viral glycoprotein (RVG) peptide on the membrane surface. We demonstrated that the aptamers were efficiently packaged into the RVG-exosomes and delivered into neurons in vitro and in vivo. Functionally, the aptamer-loaded RVG-exosomes significantly reduced the α-synuclein preformed fibril (PFF)-induced pathological aggregates, and rescued synaptic protein loss and neuronal death. Moreover, intraperitoneal administration of these exosomes into the mice with intra-striatally injected α-synuclein PFF reduced the pathological α-synuclein aggregates and improved motor impairments. In conclusion, we demonstrated that the aptamers targeting α-synuclein aggregates could be effectively delivered into the mouse brain by the RVG-exosomes and reduce the neuropathological and behavioral deficits in the mouse PD model. This study highlights the therapeutic potential of the RVG-exosome delivery of aptamer to alleviate the brain α-synuclein pathology.

Effects of different patterns of electric stimulation of the ventromedial prefrontal cortex on hippocampal-prefrontal coherence in a rat model of depression.

Deep brain stimulation (DBS) is currently being studied as a promising treatment for treatment-refractory depression. However, the optimal pattern of stimulation and the underlying mechanism remain unknown. Neuronal communication between the hippocampus and ventral medial prefrontal cortex (vmPFC) may play an important role in emotional processes relevant to depression. Here, we investigated the effect of two commonly used patterns of vmPFC stimulation: high-frequency stimulation with low intensity (HFS, 130 Hz, 100 µA) and low-frequency stimulation with high intensity (LFS, 20 Hz, 400 µA), on oscillatory network dynamics in these two depression-related brain areas, in freely-moving rats after chronic unpredictable stress (CUS). We found that one hour of either LFS or HFS of the vmPFC induced a clear antidepressant-like effect in CUS rats. Analysis of local field potential (LFP) oscillation results showed that baseline power values were lower in CUS rats than normal controls, in the beta and gamma frequency range. Both HFS and LFS produced a widespread increase in spontaneous LFP oscillations, especially beta and gamma oscillations, in vmPFC and hippocampus of control and CUS rats. Furthermore, both HFS and LFS increased coordinated activity in beta and gamma bands between these two regions in freely-moving rats. In addition, there was no difference between LFS and HFS in the antidepressant-like response and LFP oscillations. These results suggest that HFS and LFS may share a common antidepressant mechanism, in which the augmentation of beta and gamma synchrony in vmPFC and hippocampus may contribute to the improvement of depressive-like behaviors.

The Molecular Mechanism of Alpha-Synuclein Dependent Regulation of Protein Phosphatase 2A Activity.

BACKGROUND/AIMS: Alpha-synuclein (α-Syn) is a neuronal protein that is highly implicated in Parkinson's disease (PD), and protein phosphatase 2A (PP2A) is an important serine/threonine phosphatase that is associated with neurodegenerative diseases, such as PD. α-Syn can directly upregulate PP2A activity, but the underling mechanism remains unclear. Therefore, we investigated the molecular mechanism of α-Syn regulating PP2A activity. METHODS: α-Syn and its truncations were expressed in E.coli, and purified by affinity chromatography. PP2A Cα and its mutants were expressed in recombinant baculovirus, and purified by affinity chromatography combined with gel filtration chromatography. The interaction between α-Syn and PP2A Cα was detected by GST pull-down assay. PP2A activity was investigated by the colorimetric assay. RESULTS: The hydrophobic non-amyloid component (NAC) domain of α-Syn interacted with PP2A Cα and upregulated its activity. α-Syn aggregates reduced its ability to upregulate PP2A activity, since the hydrophobic domain of α-Syn was blocked during aggregation. Furthermore, in the hydrophobic center of PP2A Cα, the residue of I123 was responsible for PP2A to interact with α-Syn, and its hydrophilic mutation blocked its interaction with α-Syn as well as its activity upregulation by α-Syn. CONCLUSIONS: α-Syn bound to PP2A Cα by the hydrophobic interaction and upregulated its activity. Blocking the hydrophobic domain of α-Syn or hydrophilic mutation on the residue I123 in PP2A Cα all reduced PP2A activity upregulation by α-Syn. Overall, we explored the mechanism of α-Syn regulating PP2A activity, which might offer much insight into the basis underlying PD pathogenesis.

Novel DNA Aptamers for Parkinson's Disease Treatment Inhibit α-Synuclein Aggregation and Facilitate its Degradation.

Parkinson's disease (PD) is one of the most prevalent forms of synucleinopathies, and it is characterized neuropathologically by the presence of intracellular inclusions composed primarily of the protein α-synuclein (α-syn) in neurons. The previous immunotherapy targeting the α-syn in PD models with monoclonal antibodies has established α-syn protein as an effective target for neuronal cell death. However, due to the essential weaknesses of antibody and the unique features of aptamers, the aptamers could represent a promising alternative to the currently used antibodies in immunotherapy for PD. In this study, the purified human α-syn was used as the target for in vitro selection of aptamers using systematic evolution by exponential enrichment. This resulted in the identification of two 58-base DNA aptamers with a high binding affinity and good specificity to the α-syn, with KD values in the nanomolar range. Both aptamers could effectively reduce α-syn aggregation in vitro and in cells and target the α-syn to intracellular degradation through the lysosomal pathway. These effects consequently rescued the mitochondrial dysfunction and cellular defects caused by α-syn overexpression. To our knowledge, this is the first study to employ aptamers to block the aberrant cellular effects of the overexpressed α-syn in cells.

Photolysis of Caged-GABA Rapidly Terminates Seizures In Vivo: Concentration and Light Intensity Dependence.

The therapy of focal epilepsy remains unsatisfactory for as many as 25% of patients. The photolysis of caged-γ-aminobutyric acid (caged-GABA) represents a novel and alternative option for the treatment of intractable epilepsy. Our previous experimental results have demonstrated that the use of blue light produced by light-emitting diode to uncage ruthenium-bipyridine-triphenylphosphine-c-GABA (RuBi-GABA) can rapidly terminate paroxysmal seizure activity both in vitro and in vivo. However, the optimal concentration of RuBi-GABA, and the intensity of illumination to abort seizures, remains unknown. The aim of this study was to explore the optimal anti-seizure effects of RuBi-GABA by using implantable fibers to introduce blue light into the neocortex of a 4-aminopyridine-induced acute seizure model in rats. We then investigated the effects of different combinations of RuBi-GABA concentrations and light intensity upon seizure. Our results show that the anti-seizure effect of RuBi-GABA has obvious concentration and light intensity dependence. This is the first example of using an implantable device for the photolysis of RuBi-GABA in the therapy of neocortical seizure, and an optimal combination of RuBi-GABA concentration and light intensity was explored. These results provide important experimental data for future clinical translational studies.

Molecular basis of reactive oxygen species-induced inactivation of α4ß2 nicotinic acetylcholine receptors.

The α4ß2 neuronal nicotinic acetylcholine receptors (nAChRs) are the most widespread heteromeric nAChR subtype in the brain, mediating fast synaptic transmission. Previous studies showed that α4ß2 nAChRs could be inactivated by reactive oxygen species (ROS), but the underlying mechanism is still obscure. We found that H2O2 induced the rundown of ACh-evoked currents in human α4ß2 nAChRs and the replacement of the conserved cysteine in the M1-M2 linker of either α4 Cys245 or ß2 Cys237 with an alanine residue could prevent the current rundown. Structurally, α4 Cys245 and ß2 Cys237 are hypothesized to be in close proximity when the receptor is activated. Western blotting results showed that α4 and ß2 subunits were cross-linked when the agonist-bound receptor encountered H2O2, which could be prevented by the substitution of the conserved cysteine in the M1-M2 linker to an alanine. Thus, when agonist bound to the receptor, α4 Cys245 and ß2 Cys237 came close to each other and ROS oxidized these conserved cysteines, leading subunits to be cross-linked and trapping α4ß2 nAChRs into the inactivation state. In addition, we mimicked an experimental Parkinson's disease (PD) model in PC12 cells and found that ROS, generated by 6-hydroxydopamine (6-OHDA), could cause the current rundown in α4ß2 nAChRs, which may play a role in PD.

Functional Impact of 14 Single Nucleotide Polymorphisms Causing Missense Mutations of Human α7 Nicotinic Receptor.

The α7nicotinic receptor (nAChR) is a major subtype of the nAChRs in the central nervous system, and the receptor plays an important role in brain function. In the dbSNP database, there are 55 single nucleotide polymorphisms (SNPs) that cause missense mutations of the human α7nAChR in the coding region. In this study, we tested the impact of 14 SNPs that cause missense mutations in the agonist binding site or the coupling region between binding site and channel gate on the receptor function. The wild type or mutant receptors were expressed or co-expressed in Xenopus oocytes, and the agonist-induced currents were tested using two-electrode voltage clamp. Our results demonstrated that 6 mutants were nonfunctional, 4 mutants had reduced current expression, and 1 mutants altered ACh and nicotine efficacy in the opposite direction, and one additional mutant had slightly reduced agonist sensitivity. Interestingly, the function of most of these nonfunctional mutants could be rescued by α7nAChR positive allosteric modulator PNU-120596 and agonist-PAM 4BP-TQS. Finally, when coexpressed with the wild type, the nonfunctional mutants could also influence the receptor function. These changes of the receptor properties by the mutations could potentially have an impact on the physiological function of the α7nAChR-mediated cholinergic synaptic transmission and anti-inflammatory effects in the human SNP carriers. Rescuing the nonfunctional mutants could provide a novel way to treat the related disorders.

The coupling interface and pore domain codetermine the single-channel activity of the α7 nicotinic receptor.

Ligand-gated ion channels play a role in mediating fast synaptic transmission for communication between neurons. However, the structural basis for the functional coupling of the binding and pore domains, resulting in channel opening, remains a topic of intense investigation. Here, a series of α7 nicotinic receptor mutants were constructed for expression in cultured mammalian cells, and their single-channel properties were examined using the patch-clamp technique combined with radio ligand binding and the fluorescence staining technique. We demonstrated that the replacement of the four pore-lining residues in the channel domain of the α7 nicotinic receptor with the hydrophilic residue serine prolongs the open-channel lifetime, although the conductance of these mutants decreases. At the coupling interface between the extracellular and transmembrane domains, when the VRW residues in the Cys-loop were substituted with the corresponding residues (i.e., IYN) in the 5-HT3A receptor, the single-channel activity elicited by acetylcholine is impaired. This effect occurred despite the expression of the mutant receptors on the cell surface and despite the fact that the apparent Kd values were much lower than those of the wild-type α7 receptor. When we further lowered the channel-gating barrier of this chimera to enhance the open-channel probability, the loss of function was rescued. Overall, we explored the microscopic mechanisms underlying the interplay between the channel domains and the coupling interface that affect the channel activity, and we generated an allosteric gating model for the α7 receptor. This model shows that the gating machinery and coupling assembly codetermine the single-channel gating kinetics. These results likely apply to all channels in the Cys-loop receptor family.

Mechanisms of transient receptor potential vanilloid 1 activation and sensitization by allyl isothiocyanate.

Allyl isothiocyanate (AITC; aka, mustard oil) is a powerful irritant produced by Brassica plants as a defensive trait against herbivores and confers pungency to mustard and wasabi. AITC is widely used experimentally as an inducer of acute pain and neurogenic inflammation, which are largely mediated by the activation of nociceptive cation channels transient receptor potential ankyrin 1 and transient receptor potential vanilloid 1 (TRPV1). Although it is generally accepted that electrophilic agents activate these channels through covalent modification of cytosolic cysteine residues, the mechanism underlying TRPV1 activation by AITC remains unknown. Here we show that, surprisingly, AITC-induced activation of TRPV1 does not require interaction with cysteine residues, but is largely dependent on S513, a residue that is involved in capsaicin binding. Furthermore, AITC acts in a membrane-delimited manner and induces a shift of the voltage dependence of activation toward negative voltages, which is reminiscent of capsaicin effects. These data indicate that AITC acts through reversible interactions with the capsaicin binding site. In addition, we show that TRPV1 is a locus for cross-sensitization between AITC and acidosis in nociceptive neurons. Furthermore, we show that residue F660, which is known to determine the stimulation by low pH in human TRPV1, is also essential for the cross-sensitization of the effects of AITC and low pH. Taken together, these findings demonstrate that not all reactive electrophiles stimulate TRPV1 via cysteine modification and help understanding the molecular bases underlying the surprisingly large role of this channel as mediator of the algesic properties of AITC.

The structural mechanism of the Cys-loop receptor desensitization.

The cys-loop receptors are neurotransmitter-operated ion channels, which mediate fast synaptic transmission for communication between neurons. However, prolonged exposure to the neurotransmitter drives the receptor to a desensitization state, which plays an important role in shaping synaptic transmission. Much progress has been made through more than half a century's research since Katz and Thesleff first descried desensitization for muscle nicotinic acetylcholine receptor. In this review, we summarized recent research developments of receptor desensitization. Now, it has been identified that many parts of the receptor, such as the pore domain (including the hinge in the M2-M3 linker), the binding domain, the coupling region, and the intracellular domain, are all involved in the cys-loop receptor desensitization and that uncoupling between the amino-terminal domain and channel lining domain seems to play a central role in desensitization. This uncoupling is mainly governed by the balance between coupling strength and relative tightness of gating machinery and influenced by other parts of the receptor. Agonist binding induces conformational change to overcome the gating barrier to open the channel through the stressed coupling region, which is subsequently broken, causing receptor desensitization. With rapid advancement in structural biology of membrane receptors, final validation of this mechanism is expected to occur in the near future when the high-resolution structure of the desensitized state is available.

Desensitization of alpha7 nicotinic receptor is governed by coupling strength relative to gate tightness.

Binding of a neurotransmitter to its membrane receptor opens an integral ion conducting pore. However, prolonged exposure to the neurotransmitter drives the receptor to a refractory state termed desensitization, which plays an important role in shaping synaptic transmission. Despite intensive research in the past, the structural mechanism of desensitization is still elusive. Using mutagenesis and voltage clamp in an oocyte expression system, we provide several lines of evidence supporting a novel hypothesis that uncoupling between binding and gating machinery is the underlying mechanism for α7 nicotinic receptor (nAChR) desensitization. First, the decrease in gate tightness was highly correlated to the reduced desensitization. Second, nonfunctional mutants in three important coupling loops (loop 2, loop 7, and the M2-M3 linker) could be rescued by a gating mutant. Furthermore, the decrease in coupling strength in these rescued coupling loop mutants reversed the gating effect on desensitization. Finally, coupling between M1 and hinge region of the M2-M3 linker also influenced the receptor desensitization. Thus, the uncoupling between N-terminal domain and transmembrane domain, governed by the balance of coupling strength and gate tightness, underlies the mechanism of desensitization for the α7 nAChR.

TRPM3 is a nociceptor channel involved in the detection of noxious heat.

Transient receptor potential melastatin-3 (TRPM3) is a broadly expressed Ca(2+)-permeable nonselective cation channel. Previous work has demonstrated robust activation of TRPM3 by the neuroactive steroid pregnenolone sulfate (PS), but its in vivo gating mechanisms and functions remained poorly understood. Here, we provide evidence that TRPM3 functions as a chemo- and thermosensor in the somatosensory system. TRPM3 is molecularly and functionally expressed in a large subset of small-diameter sensory neurons from dorsal root and trigeminal ganglia, and mediates the aversive and nocifensive behavioral responses to PS. Moreover, we demonstrate that TRPM3 is steeply activated by heating and underlies heat sensitivity in a subset of sensory neurons. TRPM3-deficient mice exhibited clear deficits in their avoidance responses to noxious heat and in the development of inflammatory heat hyperalgesia. These experiments reveal an unanticipated role for TRPM3 as a thermosensitive nociceptor channel implicated in the detection of noxious heat.

Inhibition of the cation channel TRPV4 improves bladder function in mice and rats with cyclophosphamide-induced cystitis.

Reduced functional bladder capacity and concomitant increased micturition frequency (pollakisuria) are common lower urinary tract symptoms associated with conditions such as cystitis, prostatic hyperplasia, neurological disease, and overactive bladder syndrome. These symptoms can profoundly affect the quality of life of afflicted individuals, but available pharmacological treatments are often unsatisfactory. Recent work has demonstrated that the cation channel TRPV4 is highly expressed in urothelial cells and plays a role in sensing the normal filling state of the bladder. In this article, we show that the development of cystitis-induced bladder dysfunction is strongly impaired in Trpv4(-/-) mice. Moreover, we describe HC-067047, a previously uncharacterized, potent, and selective TRPV4 antagonist that increases functional bladder capacity and reduces micturition frequency in WT mice and rats with cystitis. HC-067047 did not affect bladder function in Trpv4(-/-) mice, demonstrating that its in vivo effects are on target. These results indicate that TRPV4 antagonists may provide a promising means of treating bladder dysfunction.

A novel nicotinic acetylcholine receptor subtype in basal forebrain cholinergic neurons with high sensitivity to amyloid peptides.

Nicotinic acetylcholine receptors (nAChRs) containing alpha7 subunits are thought to assemble as homomers. alpha7-nAChR function has been implicated in learning and memory, and alterations of alpha7-nAChR have been found in patients with Alzheimer's disease (AD). Here we report findings consistent with a novel, naturally occurring nAChR subtype in rodent, basal forebrain cholinergic neurons. In these cells, alpha7 subunits are coexpressed, colocalize, and coassemble with beta2 subunit(s). Compared with homomeric alpha7-nAChRs from ventral tegmental area neurons, functional, presumably heteromeric alpha7beta2-nAChRs on cholinergic neurons freshly dissociated from medial septum/diagonal band (MS/DB) exhibit relatively slow kinetics of whole-cell current responses to nicotinic agonists and are more sensitive to the beta2 subunit-containing nAChR-selective antagonist, dihydro-beta-erythroidine (DHbetaE). Interestingly, presumed, heteromeric alpha7beta2-nAChRs are highly sensitive to functional inhibition by pathologically relevant concentrations of oligomeric, but not monomeric or fibrillar, forms of amyloid beta(1-42) (Abeta(1-42)). Slow whole-cell current kinetics, sensitivity to DHbetaE, and specific antagonism by oligomeric Abeta(1-42) also are characteristics of heteromeric alpha7beta2-nAChRs, but not of homomeric alpha7-nAChRs, heterologously expressed in Xenopus oocytes. Moreover, choline-induced currents have faster kinetics and less sensitivity to Abeta when elicited from MS/DB neurons derived from nAChR beta2 subunit knock-out mice rather than from wild-type mice. The presence of novel, functional, heteromeric alpha7beta2-nAChRs on basal forebrain cholinergic neurons and their high sensitivity to blockade by low concentrations of oligomeric Abeta(1-42) suggests possible mechanisms for deficits in cholinergic signaling that could occur early in the etiopathogenesis of AD and might be targeted by disease therapies.