Biallelic null variants in PNPLA8 cause microcephaly by reducing the number of basal radial glia.

Patatin-like phospholipase domain-containing lipase 8 (PNPLA8), one of the calcium-independent phospholipase A2 enzymes, is involved in various physiological processes through the maintenance of membrane phospholipids. Biallelic variants in PNPLA8 have been associated with a range of paediatric neurodegenerative disorders. However, the phenotypic spectrum, genotype-phenotype correlations and the underlying mechanisms are poorly understood. Here, we newly identified 14 individuals from 12 unrelated families with biallelic ultra-rare variants in PNPLA8 presenting with a wide phenotypic spectrum of clinical features. Analysis of the clinical features of current and previously reported individuals (25 affected individuals across 20 families) showed that PNPLA8-related neurological diseases manifest as a continuum ranging from variable developmental and/or degenerative epileptic-dyskinetic encephalopathy to childhood-onset neurodegeneration. We found that complete loss of PNPLA8 was associated with the more profound end of the spectrum, with congenital microcephaly. Using cerebral organoids generated from human induced pluripotent stem cells, we found that loss of PNPLA8 led to developmental defects by reducing the number of basal radial glial cells and upper-layer neurons. Spatial transcriptomics revealed that loss of PNPLA8 altered the fate specification of apical radial glial cells, as reflected by the enrichment of gene sets related to the cell cycle, basal radial glial cells and neural differentiation. Neural progenitor cells lacking PNPLA8 showed a reduced amount of lysophosphatidic acid, lysophosphatidylethanolamine and phosphatidic acid. The reduced number of basal radial glial cells in patient-derived cerebral organoids was rescued, in part, by the addition of lysophosphatidic acid. Our data suggest that PNPLA8 is crucial to meet phospholipid synthetic needs and to produce abundant basal radial glial cells in human brain development.

Tuning Excited State Character in Iridium(III) Photosensitizers and Its Influence on TTA-UC.

A series of mixed ligand, photoluminescent organometallic Ir(III) complexes have been synthesized to incorporate substituted 2-phenyl-1H-naphtho[2,3-d]imidazole cyclometalating ligands. The structures of three example complexes were categorically confirmed using X-ray crystallography each sharing very similar structural traits including evidence of interligand hydrogen bond contacts that account for the shielding effects observed in the 1H NMR spectra. The structural iterations of the cyclometalated ligand provide tuning of the principal electronic transitions that determine the visible absorption and emission properties of the complexes: emission can be tuned in the visible region between 550 and 610 nm and with triplet lifetimes up to 10 µs. The nature of the emitting state varies across the series of complexes, with different admixtures of ligand-centered and metal-to-ligand charge transfer triplet levels evident. Finally, the use of the complexes as photosensitizers in triplet-triplet annihilation energy upconversion (TTA-UC) was investigated in the solution state. The study showed that the complexes possessing the longest triplet lifetimes showed good viability as photosensitizers in TTA-UC. Therefore, the use of an electron-withdrawing group on the 2-phenyl-1H-naphtho[2,3-d]imidazole ligand framework can be used to rationally promote TTA-UC using this class of complex.

Luminescent Pt(II) Complexes Using Unsymmetrical Bis(2-pyridylimino)isoindolate Analogues.

A series of ligands based upon a 1,3-diimino-isoindoline framework have been synthesized and investigated as pincer-type (N∧N∧N) chelates for Pt(II). The synthetic route allows different combinations of heterocyclic moieties (including pyridyl, thiazole, and isoquinoline) to yield new unsymmetrical ligands. Pt(L1-6)Cl complexes were obtained and characterized using a range of spectroscopic and analytical techniques: 1H and 13C NMR, IR, UV-vis and luminescence spectroscopies, elemental analyses, high-resolution mass spectrometry, electrochemistry, and one example via X-ray crystallography which showed a distorted square planar environment at Pt(II). Cyclic voltammetry on the complexes showed one irreversible oxidation between +0.75 and +1 V (attributed to Pt2+/3+ couple) and a number of ligand-based reductions; in four complexes, two fully reversible reductions were noted between -1.4 and -1.9 V. Photophysical studies showed that Pt(L1-6)Cl absorbs efficiently in the visible region through a combination of ligand-based bands and metal-to-ligand charge-transfer features at 400-550 nm, with assignments supported by DFT calculations. Excitation at 500 nm led to luminescence (studied in both solutions and solid state) in all cases with different combinations of the heterocyclic donors providing tuning of the emission wavelength around 550-678 nm.

Synthesis and luminescent properties of hetero-bimetallic and hetero-trimetallic Ru(II)/Au(I) or Ir(III)/Au(I) complexes.

A series of Ru(II) and Ir(III) based photoluminescent complexes were synthesised that incorporate an ancillary 2,2'-bipyridine ligand adorned with either one or two pendant N-methyl imidazolium groups. These complexes have been fully characterised by an array of spectroscopic and analytical techniques. One Ir(III) example was unequivocally structurally characterised in the solid state using single crystal X-ray diffraction confirming the proposed formulation and coordination sphere. These complexes were then transformed into their heterometallic, Au(I)-containing, analogues in two steps to yield either bi- or trimetallic complexes that integrate {Au(PPh3)}+ units. X-ray diffraction was used to corroborate the solid state structure of the hetero bimetallic complex, based upon a Ru(II)-Au(I) species. The heterometallic complexes all displayed red photoluminescent features (λem = 616-629 nm) that were consistent with the parent Ru(II) or Ir(III) lumophores in each case. The modulation of the emission from the Ru(II)-Au(I) complexes was much more strongly evident than for the Ir(III)-Au(I) analogues, which is ascribed to the inherent differences in the specific triplet excited state character of the emitting states within each heterometallic species.

Alkyl chain functionalised Ir(iii) complexes: synthesis, properties and behaviour as emissive dopants in microemulsions.

Six iridium(iii) complexes of the general form [Ir(C^N)2(N^N)]X (where C^N = cyclometalating ligand; N^N = disubstituted 2,2'-bipyridine), and incorporating alkyl chains of differing lengths (C8, C10, C12), have been synthesised and characterised. The complexes have been characterised using a variety of methods including spectroscopies (NMR, IR, UV-Vis, luminescence) and analytical techniques (high resolution mass spectrometry, cyclic voltammetry, X-ray diffraction). Two dodecyl-functionalised complexes were studied for their behaviour in aqueous solutions. Although the complexes did not possess sufficient solubility to determine their critical micelle concentrations (CMC) in water, they were amenable for use as emissive dopants in a N-methyl C12 substituted imidazolium salt microemulsion carrier system with a CMC = 36.5 mM. The investigation showed that the metal doped microemulsions had increased CMCs of 40.4 and 51.3 mM and luminescent properties characterised by the dopant.

Polycationic Ru(II) Luminophores: Syntheses, Photophysics, and Application in Electrostatically Driven Sensitization of Lanthanide Luminescence.

A series of photoluminescent Ru(II) polypyridine complexes have been synthesized in a manner that varies the extent of the cationic charge. Two ligand systems (L1 and L2), based upon 2,2'-bipyridine (bipy) mono- or difunctionalized at the 5- or 5,5'-positions using N-methylimidazolium groups, were utilized. The resulting Ru(II) species therefore carried +3, +4, +6, and +8 complex moieties based on a [Ru(bipy)3]2+ core. Tetra-cationic [Ru(bipy)2(L2)][PF6]4 was characterized using XRD, revealing H-bonding interactions between two of the counteranions and the cationic unit. The ground-state features of the complexes were found to closely resemble those of the parent unfunctionalized [Ru(bipy)3]2+ complex. In contrast, the excited state properties produce a variation in emission maxima, including a bathochromic 44 nm shift of the 3MLCT band for the tetra-cationic complex; interestingly, further increases in overall charge to +6 and +8 produced a hypsochromic shift in the 3MLCT band. Supporting DFT calculations suggest that the trend in emission behavior may, in part, be due to the precise nature of the LUMO and its localization. The utility of a photoactive polycationic Ru(II) complex was then demonstrated through the sensitization of a polyanionic Yb(III) complex in free solution. The study shows that electrostatically driven ion pairing is sufficient to facilitate energy transfer between the 3MLCT donor state of the Ru(II) complex and the accepting 2F5/2 excited state of Yb(III).

2-(Thienyl)quinoxaline derivatives and their application in Ir(III) complexes yielding tuneable deep red emitters.

The synthesis and characterisation of eleven different 2-(thienyl)quinoxaline species that incorporate different points of functionality, including at the thiophene or quinoxaline rings, are described. These species display variable fluorescence properties in the visible region (λem = 401-491 nm) depending upon the molecular structures and extent of conjugation. The series of 2-(thienyl)quinoxaline species were then investigated as cyclometalating agents for Ir(III) to yield [Ir(C^N)2(bipy)]PF6 (where C^N = the cyclometalated ligand; bipy = 2,2'-bipyridine). Eight complexes were successfully isolated and fully characterised by an array of spectroscopic and analytical techniques. Two Ir(III) examples were structurally characterised in the solid state using single crystal X-ray diffraction; both structures confirmed the proposed formulations and coordination spheres in each case showing that the thiophene coordinates via a Ir-C bond. The photophysical properties of the complexes revealed that each complex is luminescent under ambient conditions with a range of emission wavelengths observed (665-751 nm) indicating that electronic tuning can be achieved via both the thienyl and quinoxaline moieties.

Organometallic Platinum(II) Photosensitisers that Demonstrate Ligand-Modulated Triplet-Triplet Annihilation Energy Upconversion Efficiencies.

A series of 2-phenylquinoxaline ligands have been synthesised that introduce either CF3 or OCF3 electron-withdrawing groups at different positions of the phenyl ring. These ligands were investigated as cyclometalating reagents for platinum(II) to give neutral complexes of the form [Pt(C^N)(acac)] (in which C^N=cyclometalating ligand; acac=acetyl acetonate). X-ray crystallographic studies on three examples showed that the complexes adopt an approximate square planar geometry. All examples revealed strong Pt-Pt linear contacts of 3.2041(6), 3.2199(3) and 3.2586(2) Å. The highly coloured complexes display efficient visible absorption at 400-500 nm (ϵ ≈5000 M-1  cm-1 ) and orange red photoluminescent characteristics (λem =603-620 nm; Φem ≤37 %), which were subtly tuned by the ligand. Triplet emitting character was confirmed by microsecond luminescence lifetimes and the photogeneration of singlet oxygen with quantum efficiencies up to 57 %. Each complex was investigated as a photosensitiser for triplet-triplet annihilation energy upconversion using 9,10-diphenylanthracene as the annihilator species: a range of good upconversion efficiencies (ΦUC 5.9-14.1 %) were observed and shown to be strongly influenced by the ligand structure in each case.

Long-lived, near-IR emission from Cr(III) under ambient conditions.

Bis-terdentate (N^N^N) ligands coordinated to Cr(III) yield complexes that display near-IR emission under aerated solvent conditions at room temperature.

N,N'-Substituted thioureas and their metal complexes: syntheses, structures and electronic properties.

The synthesis of six N,N'-substituted thiourea ligands (L1a-L3b) was achieved in two steps. A corresponding extensive series of Cu(I), Cu(II), Ni(II) and Zn(II) complexes (1-24) with varying formulations were synthesised from these ligands by the reaction of a 1 : 1 or a 1 : 2 mixture of Cu(II), Ni(II) and Zn(II) perchlorate or chloride salts. Complexes 1-24 have been comprehensively characterised by mass spectrometry, elemental analysis, UV-vis., IR, and 1H and 13C{1H} NMR spectroscopies where applicable. The X-ray crystal structures were obtained for eight examples: [(L1a)2Cu]ClO4 (1), [(L1c)2Zn](ClO4)2 (4), [(L2a)2Cu]ClO4 (6), [(L2c)2Ni](ClO4)2 (7), [(L1b)2Cu](ClO4) (15), [(L1b)CuCl] (16), [(L4)2CuCl2] (19) and [(L3b)CuClO4] (21). These studies reveal that L1c and L2c represent ligands that have undergone cleavage during reaction with the metal salt; L4 represents an intramolecular rearrangement (via a Hugershoff reaction) of L2b; and in most cases Cu(II) is reduced to Cu(I) during the ligand reaction. The X-ray crystal structures also reveal that 1, 4, 6, 15 and 16 are monometallic species in the solid state; that Cu(I) in 1, 6, 15 and 16 and Zn(II) in 4 are arranged in a distorted tetrahedral geometry; that Cu(I) in 21 adopts a trigonal planar geometry; and that in 7 and 19 the Ni(II) and Cu(II) centres, respectively, possess square planar geometry. Preliminary studies on the biological activity (using the Malaria Sybr Green I Fluorescence assay) of the thiourea containing complexes suggests that the d10 complexes, and increased ligand stoichiometries, may afford higher potency.

Advances in methods to analyse cardiolipin and their clinical applications.

Cardiolipin (CL) is a mitochondria-exclusive phospholipid, primarily localised within the inner mitochondrial membrane, that plays an essential role in mitochondrial architecture and function. Aberrant CL content, structure, and localisation have all been linked to impaired mitochondrial activity and are observed in the pathophysiology of cancer and neurological, cardiovascular, and metabolic disorders. The detection, quantification, and localisation of CL species is a valuable tool to investigate mitochondrial dysfunction and the pathophysiological mechanisms underpinning several human disorders. CL is measured using liquid chromatography, usually combined with mass spectrometry, mass spectrometry imaging, shotgun lipidomics, ion mobility spectrometry, fluorometry, and radiolabelling. This review summarises available methods to analyse CL, with a particular focus on modern mass spectrometry, and evaluates their advantages and limitations. We provide guidance aimed at selecting the most appropriate technique, or combination of techniques, when analysing CL in different model systems, and highlight the clinical contexts in which measuring CL is relevant.

Polysubstituted Ligand Framework for Color Tuning Phosphorescent Iridium(III) Complexes.

A series of ligands have been synthesized based upon a polysubstituted 2-phenylquinoxaline core structure. These ligands introduce different combinations of fluorine and methyl substituents on both the phenyl and quinoxaline constituent rings. The resultant investigation of these species as cyclometalating agents for Ir(III) gave cationic complexes of the form [Ir(C^N)2(bipy)]PF6 (where C^N = cyclometalating ligand; bipy = 2,2'-bipyridine). X-ray crystallographic studies were conducted on four complexes and each revealed the expected distorted octahedral geometry based upon a cis-C,C and trans-N,N ligand arrangement at Ir(III). Supporting computational studies predict that each of the complexes share the same general descriptions for the frontier orbitals. TD-DFT calculations suggest MLCT contributions to the lowest energy absorption and a likely MLCT/ILCT/LLCT nature to the emitting state. Experimentally, the complexes display tunable luminescence across the yellow-orange-red part of the visible spectrum (λem = 579-655 nm).

Monitoring spin-crossover phenomena via Re(I) luminescence in hybrid Fe(II) silica coated nanoparticles.

Bare (1) and silica coated (1@SiO2) spin crossover (SCO) nanoparticles based on the polymer {[Fe(NH2Trz)3](BF4)2}n have been prepared following a water-in-oil synthetic procedure. For 1, the critical temperatures of the spin transition are TC↓ = 214.6 K and TC↑ = 220.9 K. For 1@SiO2, the abruptness of the transition is enhanced and the critical temperatures are centred at room temperature (TC↓ = 292.1 K and TC↑ = 296.3 K). An inert Re(I) complex of formula [Re(phen)(CO)3(PETES)](PF6) (phen = 1, 10-phenanthroline; PETES = 2(4-pyridylethyl)triethoxysilane) (Re) was also synthesized yielding intense green emission centred at λem = 560 nm. The grafting of this complex on the silica shell of 1@SiO2 led to a bifunctional SCO-luminescence composite (1@SiO2/Re) whose luminescence properties were tuned by the spin state switching. Temperature-variable photophysical studies showed that luminescence and spin transition were synchronized through a radiative (trivial) energy transfer mechanism between the Re(I) and the Fe(II)-LS (LS, Low Spin) centres.

Gene therapy restores dopamine transporter expression and ameliorates pathology in iPSC and mouse models of infantile parkinsonism.

Most inherited neurodegenerative disorders are incurable, and often only palliative treatment is available. Precision medicine has great potential to address this unmet clinical need. We explored this paradigm in dopamine transporter deficiency syndrome (DTDS), caused by biallelic loss-of-function mutations in SLC6A3, encoding the dopamine transporter (DAT). Patients present with early infantile hyperkinesia, severe progressive childhood parkinsonism, and raised cerebrospinal fluid dopamine metabolites. The absence of effective treatments and relentless disease course frequently leads to death in childhood. Using patient-derived induced pluripotent stem cells (iPSCs), we generated a midbrain dopaminergic (mDA) neuron model of DTDS that exhibited marked impairment of DAT activity, apoptotic neurodegeneration associated with TNFα-mediated inflammation, and dopamine toxicity. Partial restoration of DAT activity by the pharmacochaperone pifithrin-µ was mutation-specific. In contrast, lentiviral gene transfer of wild-type human SLC6A3 complementary DNA restored DAT activity and prevented neurodegeneration in all patient-derived mDA lines. To progress toward clinical translation, we used the knockout mouse model of DTDS that recapitulates human disease, exhibiting parkinsonism features, including tremor, bradykinesia, and premature death. Neonatal intracerebroventricular injection of human SLC6A3 using an adeno-associated virus (AAV) vector provided neuronal expression of human DAT, which ameliorated motor phenotype, life span, and neuronal survival in the substantia nigra and striatum, although off-target neurotoxic effects were seen at higher dosage. These were avoided with stereotactic delivery of AAV2.SLC6A3 gene therapy targeted to the midbrain of adult knockout mice, which rescued both motor phenotype and neurodegeneration, suggesting that targeted AAV gene therapy might be effective for patients with DTDS.

Aromatic l-amino acid decarboxylase deficiency: a patient-derived neuronal model for precision therapies.

Aromatic l-amino acid decarboxylase (AADC) deficiency is a complex inherited neurological disorder of monoamine synthesis which results in dopamine and serotonin deficiency. The majority of affected individuals have variable, though often severe cognitive and motor delay, with a complex movement disorder and high risk of premature mortality. For most, standard pharmacological treatment provides only limited clinical benefit. Promising gene therapy approaches are emerging, though may not be either suitable or easily accessible for all patients. To characterize the underlying disease pathophysiology and guide precision therapies, we generated a patient-derived midbrain dopaminergic neuronal model of AADC deficiency from induced pluripotent stem cells. The neuronal model recapitulates key disease features, including absent AADC enzyme activity and dysregulated dopamine metabolism. We observed developmental defects affecting synaptic maturation and neuronal electrical properties, which were improved by lentiviral gene therapy. Bioinformatic and biochemical analyses on recombinant AADC predicted that the activity of one variant could be improved by l-3,4-dihydroxyphenylalanine (l-DOPA) administration; this hypothesis was corroborated in the patient-derived neuronal model, where l-DOPA treatment leads to amelioration of dopamine metabolites. Our study has shown that patient-derived disease modelling provides further insight into the neurodevelopmental sequelae of AADC deficiency, as well as a robust platform to investigate and develop personalized therapeutic approaches.

Bis-cyclometalated iridium(iii) complexes with terpyridine analogues: syntheses, structures, spectroscopy and computational studies.

Two ligands based upon a 2,6-disubstituted pyridine bridge introduce bis-quinoxalinyl units in a fashion that yields analogues to the archetypal terdentate ligand, 2,2':6',2''-terpyridine. The ligands were synthesised from the key intermediate 2,6-bis(bromoacetyl)pyridine: a new, high-yielding route is described for this reagent. Two ligand variants (differentiated by H/Me substituents on the quinoxaline ring) were explored as coordinating moieties for iridium(iii) in the development of luminescent complexes. Computational studies (DFT approaches employing B3LYP, B3LYP/LANL2DZ, and M062X/LANL2DZ levels) were used to investigate the geometric and coordination mode preferences of the new ligands and two possibilities arose from theoretical investigations: [Ir(N^N^N)2]3+ and [Ir(N^N^C)2]+, with the former predicted to be more energetically favourable. Upon synthesis and isolation of the Ir(iii) complexes, X-ray crystallographic studies revealed coordination spheres that were cyclometalated, the structures both showing a [Ir(N^N^C)2]PF6 arrangement. Further spectroscopic characterization via NMR confirmed the ligand arrangements in the complexes, and photophysical studies, supported by DFT, showed that a mixture of metal-to-ligand charge transfer (MLCT) and intra-ligand charge transfer (ILCT) character is likely to contribute to the emission features of the complexes, which phosphoresce orange-red (λ em = 580-618 nm). The emission wavelength was influenced by the substituents on the quinoxaline ring (H vs. Me), thereby implying further tuneability is possible with future ligand iterations.

Iridium(III) Sensitisers and Energy Upconversion: The Influence of Ligand Structure upon TTA-UC Performance.

Six substituted ligands based upon 2-(naphthalen-1-yl)quinoline-4-carboxylate and 2-(naphthalen-2-yl)quinoline-4-carboxylate have been synthesised in two steps from a range of commercially available isatin derivatives. These species are shown to be effective cyclometallating ligands for IrIII , yielding complexes of the form [Ir(C^N)2 (bipy)]PF6 (where C^N=cyclometallating ligand; bipy=2,2'-bipyridine). X-ray crystallographic studies on three examples demonstrate that the complexes adopt a distorted octahedral geometry wherein a cis-C,C and trans-N,N coordination mode is observed. Intraligand torsional distortions are evident in all cases. The IrIII complexes display photoluminescence in the red part of the visible region (668-693 nm), which is modestly tuneable through the ligand structure. The triplet lifetimes of the complexes are clearly influenced by the precise structure of the ligand in each case. Supporting computational (DFT) studies suggest that the differences in observed triplet lifetime are likely due to differing admixtures of ligand-centred versus MLCT character instilled by the facets of the ligand structure. Triplet-triplet annihilation upconversion (TTA-UC) measurements demonstrate that the complexes based upon the 1-naphthyl derived ligands are viable photosensitisers with upconversion quantum efficiencies of 1.6-6.7 %.

Correction to: Consensus guideline for the diagnosis and treatment of tetrahydrobiopterin (BH4) deficiencies.

An amendment to this paper has been published and can be accessed via the original article.