Neuronal Storage Research Articles

Dopamine-mediated interactions between short- and long-term memory dynamics

In dynamic environments, animals make behavioural decisions on the basis of the innate valences of sensory cues and information learnt about these cues across multiple timescales1–3. However, it remains unclear how the innate valence of a sensory stimulus affects the acquisition of learnt valence information and subsequent memory dynamics. Here we show that in the Drosophila brain, interconnected short- and long-term memory units of the mushroom body jointly regulate memory through dopamine signals that encode innate and learnt sensory valences. By performing time-lapse in vivo voltage-imaging studies of neural spiking in more than 500 flies undergoing olfactory associative conditioning, we found that protocerebral posterior lateral 1 dopamine neurons (PPL1-DANs)4 heterogeneously and bidirectionally encode innate and learnt valences of punishment, reward and odour cues. During learning, these valence signals regulate memory storage and extinction in mushroom body output neurons (MBONs)5. During initial conditioning bouts, PPL1-γ1pedc and PPL1-γ2α′1 neurons control short-term memory formation, which weakens inhibitory feedback from MBON-γ1pedc>α/β to PPL1-α′2α2 and PPL1-α3. During further conditioning, this diminished feedback allows these two PPL1-DANs to encode the net innate plus learnt valence of the conditioned odour cue, which gates long-term memory formation. A computational model constrained by the fly connectome6,7 and our spiking data explains how dopamine signals mediate the circuit interactions between short- and long-term memory traces, yielding predictions that our experiments confirmed. Overall, the mushroom body achieves flexible learning through the integration of innate and learnt valences in parallel learning units sharing feedback interconnections. This hybrid physiological–anatomical mechanism may be a general means by which dopamine regulates memory dynamics in other species and brain structures, including the vertebrate basal ganglia.

Gain-of-function variants in CLCN7 cause hypopigmentation and lysosomal storage disease

Together with its β-subunit OSTM1, ClC-7 performs 2Cl−/H+ exchange across lysosomal membranes. Pathogenic variants in either gene cause lysosome-related pathologies, including osteopetrosis, lysosomal storage, and pigmentation defects. CLCN7 variants can cause recessive or dominant disease. Different variants entail different sets of symptoms. Loss of ClC-7 causes osteopetrosis and mostly neuronal lysosomal storage. A recently reported de novo CLCN7 mutation (p.Tyr715Cys) causes widespread severe lysosome pathology and hypopigmentation (‘HOD syndrome’), but no osteopetrosis. We now describe two additional HOD individuals with the previously described p.Tyr715Cys and a novel p.Lys285Thr mutation, respectively. Both mutations decreased ClC-7 inhibition by PI(3,5)P2 and affected residues lining its binding pocket, and shifted voltage-dependent gating to less positive potentials, an effect partially conferred to WT subunits in WT/mutant heteromers. This shift predicts augmented pH gradient-driven Cl− uptake into vesicles. Overexpressing either mutant induced large lysosome-related vacuoles. This effect depended on Cl−/H+-exchange, as shown using mutants carrying uncoupling mutations. Fibroblasts from the p.Y715C patient also displayed giant vacuoles. This was not observed with p.K285T fibroblasts probably due to some ClC-7K285T-retained PI(3,5)P2 sensitivity. The gain of function caused by the shifted voltage-dependence of either mutant likely is the main cause of their pathogenicity. Their loss of PI(3,5)P2 inhibition will further increase currents, but may not be a general feature of HOD. Overactivity of ClC-7 induces pathologically enlarged vacuoles in many tissues, which is distinct from lysosomal storage observed with the loss of ClC-7 function. Osteopetrosis results from a loss of ClC-7, but osteoclasts remain resilient to increased ClC-7 activity.

Historical review: The golden age of the Golgi method in human neuropathology.

Golgi methods were used to study human neuropathology in the 1970s, 1980s, and 1990s of the last century. Although a relatively small number of laboratories applied these methods, their impact was crucial by increasing knowledge about: (1) the morphology, orientation, and localization of neurons in human cerebral and cerebellar malformations and ganglionic tumors, and (2) the presence of abnormal structures including large and thin spines (spine dysgenesis) in several disorders linked to mental retardation, focal enlargements of the axon hillock and dendrites (meganeurites) in neuronal storage diseases, growth cone-like appendages in Alzheimer disease, as well as abnormal structures in other dementias. Although there were initial concerns about their reliability, reduced dendritic branches and dendritic spines were identified as common alterations in mental retardation, dementia, and other pathological conditions. Similar observations in appropriate experimental models have supported many abnormalities that were first identified using Golgi methods in human material. Moreover, electron microscopy, immunohistochemistry, fluorescent tracers, and combined methods have proven the accuracy of pioneering observations uniquely visualized as 3D images of fully stained individual neurons. Although Golgi methods had their golden age many years ago, these methods may still be useful complementary tools in human neuropathology.

Cellular and Molecular Mechanisms Underlying Synaptic Subcellular Specificity.

Chemical synapses are essential for neuronal information storage and relay. The synaptic signal received or sent from spatially distinct subcellular compartments often generates different outcomes due to the distance or physical property difference. Therefore, the final output of postsynaptic neurons is determined not only by the type and intensity of synaptic inputs but also by the synaptic subcellular location. How synaptic subcellular specificity is determined has long been the focus of study in the neurodevelopment field. Genetic studies from invertebrates such as Caenorhabditis elegans (C. elegans) have uncovered important molecular and cellular mechanisms required for subcellular specificity. Interestingly, similar molecular mechanisms were found in the mammalian cerebellum, hippocampus, and cerebral cortex. This review summarizes the comprehensive advances in the cellular and molecular mechanisms underlying synaptic subcellular specificity, focusing on studies from C. elegans and rodents.

Transport and inhibition mechanism for VMAT2-mediated synaptic vesicle loading of monoamines.

Monoamine neurotransmitters such as serotonin and dopamine are loaded by vesicular monoamine transporter 2 (VMAT2) into synaptic vesicles for storage and subsequent release in neurons. Impaired VMAT2 function underlies various neuropsychiatric diseases. VMAT2 inhibitors reserpine and tetrabenazine are used to treat hypertension, movement disorders associated with Huntington's Disease and Tardive Dyskinesia. Despite its physiological and pharmacological significance, the structural basis underlying VMAT2 substrate recognition and its inhibition by various inhibitors remains unknown. Here we present cryo-EM structures of human apo VMAT2 in addition to states bound to serotonin, tetrabenazine, and reserpine. These structures collectively capture three states, namely the lumen-facing, occluded, and cytosol-facing conformations. Notably, tetrabenazine induces a substantial rearrangement of TM2 and TM7, extending beyond the typical rocker-switch movement. These functionally dynamic snapshots, complemented by biochemical analysis, unveil the essential components responsible for ligand recognition, elucidate the proton-driven exchange cycle, and provide a framework to design improved pharmaceutics targeting VMAT2.

SCA34 caused by ELOVL4 L168F mutation is a lysosomal lipid storage disease sharing pathology features with neuronal ceroid lipofuscinosis and peroxisomal disorders.

Spinocerebellar ataxia 34 (SCA34) is a late-onset progressive ataxia caused by a mutation in ELOVL4, a gene involved in the biosynthesis of very long-chain fatty acids (VLCFAs). We performed post-mortem neuropathological examinations on four SCA34 patients with the ELOVL4 L168F mutation and compared the findings to age-matched controls. Specific gross findings of SCA34 were limited to pontocerebellar atrophy. On light microscopy, pontine base showed neuronal loss and storage of an autofluorescent lipopigment positive on oil red O, PAS and Hale's colloidal iron and negative on Alcian blue and Luxol fast blue (LFB). Among the swollen neurons were abundant CD68+ /CD163+ /IBA1- macrophages laden with a material with similar histochemical profile as in neurons except for the lack of autofluorescence and oil red O positivity and the presence of needle-like birefringent inclusions. Normal resting IBA1 + microglia were generally absent from pontine base nuclei but present in normal numbers elsewhere in the pons. In dentate nucleus neurons, atrophy was milder than in the pontine base and the coarser storage material was LFB-positive, closely resembling lipofuscin. On electron microscopy, dentate nucleus neurons showed neuronal storage of tridimensionally organized trilaminar spicules within otherwise normal lipofuscin, while in the more affected pontine base neurons, lipofuscin was almost completely replaced by the storage material. Storage macrophages were tightly packed with stacks of unorganized trilaminar spicules, reminiscent of the storage material seen in peroxisomal disorders and thought to represent VLCFAs incorporated in complex polar lipids. In summary, we provide histochemical and ultrastructural evidence that SCA34 is a lipid storage disease, the first among the currently known SCAs, and that the storage lipid is accumulating within neuronal lipofuscin. Our findings suggest that the storage lipid is similar to the one accumulating in non-neuronal cells in peroxisomal disorders and provide the first ultrastructural description of this type of material within neurons.

Glucosamine amends CNS pathology in mucopolysaccharidosis IIIC mouse expressing misfolded HGSNAT.

The majority of mucopolysaccharidosis IIIC (MPS IIIC) patients have missense variants causing misfolding of heparan sulfate acetyl-CoA:α-glucosaminide N-acetyltransferase (HGSNAT), which are potentially treatable with pharmacological chaperones. To test this approach, we generated a novel HgsnatP304L mouse model expressing misfolded HGSNAT Pro304Leu variant. HgsnatP304L mice present deficits in short-term and working/spatial memory 2-4 mo earlier than previously described constitutive knockout Hgsnat-Geo mice. HgsnatP304L mice also show augmented severity of neuroimmune response, synaptic deficits, and neuronal storage of misfolded proteins and gangliosides compared with Hgsnat-Geo mice. Expression of misfolded human Pro311Leu HGSNAT protein in cultured hippocampal Hgsnat-Geo neurons further reduced levels of synaptic proteins. Memory deficits and majority of brain pathology were rescued in mice receiving HGSNAT chaperone, glucosamine. Our data for the first time demonstrate dominant-negative effects of misfolded HGSNAT Pro304Leu variant and show that they are treatable by oral administration of glucosamine. This suggests that patients affected with mutations preventing normal folding of the enzyme can benefit from chaperone therapy.

Complementary Role of Combined Indirect and Direct Cardiac Sympathetic (Hyper)Activity Assessment in Patients with Heart Failure by Spectral Analysis of Heart Rate Variability and Nuclear Imaging: Possible Application in the Evaluation of Exercise Training Effects.

In chronic heart failure (CHF), abnormalities in cardiac autonomic control, characterized by sympathetic overactivity, contribute to the progression of the disease and are associated with an unfavorable prognosis. Assessing cardiac autonomic status is clinically important in the management of patients with CHF. To this aim, heart rate variability (HRV) analysis has been extensively used as a non-invasive tool for assessing cardiac autonomic regulation, and has been shown to predict the clinical outcome in patients with CHF. Adrenergic nerve activity has also been estimated using iodine-123 (I-123) metaiodobenzylguanidine (MIBG), a noradrenaline analogue. MIBG is an analogue of norepinephrine sharing the same cellular mechanism of uptake, storage, and release in presynaptic sympathetic neurons. As an innervation tracer, 123I-MIBG allows for the evaluation of cardiac sympathetic neuronal function. Cardiac MIBG imaging has also been reported to predict a poor clinical outcome in CHF. MIBG provides direct information on the function of the presynaptic sympathetic nerve endings, whereas HRV, which depends on postsynaptic signal transduction, reflects the end-organ response of the sinus node. The aim of this brief review is to provide the reader with some basic concepts regarding the spectral analysis of HRV and MIBG, highlighting what is known about their respective roles in detecting cardiac sympathetic hyperactivity in CHF and, in perspective, their possible combined use in assessing non-pharmacological treatments in patients with CHF and reduced ejection fraction, with a particular focus on the effects of exercise training.

Neuropathology of Pediatric SARS-CoV-2 Infection in the Forensic Setting: Novel Application of Ex Vivo Imaging in Analysis of Brain Microvasculature.

Two years into the COVID-19 pandemic, there are few published accounts of postmortem SARS-CoV-2 pathology in children. We report 8 such cases (4 infants aged 7–36 weeks, 4 children aged 5–15 years). Four underwent ex vivo magnetic resonance neuroimaging, to assist in identification of subtle lesions related to vascular compromise. All infants were found unresponsive (3 in unsafe sleeping conditions); all but 1 had recent rhinitis and/or influenza-like illness (ILI) in the family; 1 had history of sickle cell disease. Ex vivo neuroimaging in 1 case revealed white matter (WM) signal hyperintensity and diffuse exaggeration of perivascular spaces, corresponding microscopically to WM mineralization. Neurohistology in the remaining 3 infants variably encompassed WM gliosis and mineralization; brainstem gliosis; perivascular vacuolization; perivascular lymphocytes and brainstem microglia. One had ectopic hippocampal neurons (with pathogenic variant in DEPDC5). Among the children, 3 had underlying conditions (e.g., obesity, metabolic disease, autism) and all presented with ILI. Three had laboratory testing suggesting multisystem inflammatory syndrome (MIS-C). Two were hospitalized for critical care including mechanical ventilation and extracorporeal membrane oxygenation (ECMO); one (co-infected with adenovirus) developed right carotid stroke ipsilateral to the ECMO cannula and the other required surgery for an ingested foreign body. Autopsy findings included: acute lung injury in 3 (1 with microthrombi); and one each with diabetic ketoacidosis and cardiac hypertrophy; coronary and cerebral arteritis and aortitis, resembling Kawasaki disease; and neuronal storage and enlarged fatty liver. All 4 children had subtle meningoencephalitis, focally involving the brainstem. On ex vivo neuroimaging, 1 had focal pontine susceptibility with corresponding perivascular inflammation/expanded perivascular spaces on histopathology. Results suggest SARS-CoV-2 in infants may present as sudden unexpected infant death, while in older children, signs and symptoms point to severe disease. Underlying conditions may predispose to fatal outcomes. As in adults, the neuropathologic changes may be subtle, with vascular changes such as perivascular vacuolization and gliosis alongside sparse perivascular lymphocytes. Detection of subtle vascular pathology is enhanced by ex vivo neuroimaging. Additional analysis of the peripheral/autonomic nervous system and investigation of co-infection in children with COVID-19 is necessary to understand risk for cardiovascular collapse/sudden death.

Hypoglossal Motoneuron (XII MN) Survival Over the Lifespan in Pompe Disease

Glycogen Storage Disease II, more commonly known as Pompe disease, is a lysosomal storage disease characterized by a partial or complete loss of acid α‐glucosidase (GAA) activity. Lingual impairment and sleep disordered breathing are common symptoms of Pompe disease with a potential role of XII MN dysfunction in both cases. Through innervation of the tongue muscles, XII MNs contribute to swallowing, speech, and maintenance of upper airway patency. Studies of Pompe (Gaa‐/‐) mice have found molecular and histological markers of neurodegeneration, and a recent case study of a patient with late‐onset Pompe disease revealed atrophied and possible degenerating XII MNs. However, whether the absence of GAA activity in Pompe disease directly leads to death of XII MNs is not definitively established. Because the preponderance of evidence indicates progressive neurodegeneration in Pompe disease, here we tested the hypothesis that when XII MNs do not express GAA activity, there will be a progressive loss of these cells over the lifespan. A novel transgenic mouse model (ChAT‐cre+/+●Gaa+/‐●eYFP+/+●Flox‐Gaa+/+) was generated in which GAA activity is absent in ChAT positive neurons, which also carry a yellow fluorescent protein (YFP) marker. Thus MNs in this mouse model do not express GAA, but skeletal myofibers have normal GAA expression. Brainstem tissue was harvested in young (1‐3 months of age) and advanced age mice (21‐24 months). Tissues were cryosectioned at 20 μm increments across the entire XII motor nucleus. Sections were slide mounted and visualized under fluorescence microscopy to detect YFP. The XII motor nucleus was identified based on anatomical landmarks and MNs were identified based on location, size, morphology, and YFP fluorescence. Preliminary MN counts across the rostral‐caudal extent of the XII nucleus (n=3 per group) indicate an approximately 7% decline in detectable YFP‐positive XII MNs over the age span (young: 658±19; old 617±28). Further, the histological appearance of XII MNs showed striking differences between the two age groups. The MNs from older mice showed the prototypical hallmarks of neuronal lysosomal storage disease including a markedly enlarged soma and extensive vacuolization. These preliminary findings are consistent with the hypothesis that absence of GAA in XII MNs leads to a histopathological appearance and neurodegeneration. Ongoing studies are evaluating XII MNs in age matched wild type mice.

Voltage-Driven Alterations to Neuron Viscoelasticity.

The consideration of neurons as coupled mechanical-electrophysiological systems is supported by a growing body of experimental evidence, including observations that cell membranes mechanically deform during the propagation of an action potential. However, the short-term (seconds to minutes) influence of membrane voltage on the mechanical properties of a neuron at the single-cell level remains unknown. Here, we use microscale dynamic mechanical analysis to demonstrate that changes in membrane potential induce changes in the mechanical properties of individual neurons. We simultaneously measured the membrane potential and mechanical properties of individual neurons through a multiphysics single-cell setup. Membrane voltage of a single neuron was measured through whole-cell patch clamp. The mechanical properties of the same neuron were measured through a nanoindenter, which applied a dynamic indentation to the neuron at different frequencies. Neuronal storage and loss moduli were lower for positive voltages than negative voltages. The observed effects of membrane voltage on neuron mechanics could be due to piezoelectric or flexoelectric effects and altered ion distributions under the applied voltage. Such effects could change cell mechanics by changing the intermolecular interactions between ions and the various biomolecules within the membrane and cytoskeleton.

Dysfunction of vesicular storage in young-onset Parkinson's patient-derived dopaminergic neurons and organoids revealed by single cell electrochemical cytometry.

Electrochemical cytometry based on nano-tip microelectrodes was used to quantify the vesicular storage at the single-cell level in human neurons and midbrain organoids which acted as disease models of young-onset Parkinson's disease (YOPD). Human dopaminergic (DA) neurons and midbrain organoids were derived from an induced pluripotent stem cell line from one YOPD patient. We show a significant deficiency in vesicular catecholamine storage and a slower pore forming process on the surface of the microelectrode in the DA neurons derived from the YOPD patient. The upregulation of α-synuclein in both neurons and organoids derived from the YOPD patient is associated with vesicular storage dysfunction, revealing a correlation between the pathogenesis of YOPD and vesicular chemical storage deficiency, a novel chemical insight into the potential pathology of YOPD. Notably, efficacy evaluation and drug testing were performed with our platform to demonstrate that both amantadine, a clinical drug for Parkinson's disease (PD), and phorbol 12-myristate 13-acetate, an attractive candidate, ameliorate the dysfunction of vesicular storage in DA neurons derived from the YOPD patient. Our platform offers promising avenues for new drug discovery for PD and other neurodegenerative disorders.

White Matter Pathology as a Barrier to Gangliosidosis Gene Therapy.

The gangliosidoses are a family of neurodegenerative lysosomal storage diseases that have recently seen promising advances in gene therapy. White matter deficits are well established components of gangliosidosis pathology that are now receiving more attention because they are partially refractory to correction by gene therapy. After a brief synopsis of normal myelinogenesis, this review outlines current viewpoints on the origin of white matter deficits in the gangliosidoses and potential obstacles to treating them effectively by gene therapy. Dysmyelinogenesis (failure of myelin sheaths to form properly) is proposed as the predominant contributor to white matter pathology, but precise mechanistic details are not well understood. The involvement of neuronal storage deficits may extend beyond secondary demyelination (destruction of myelin due to axonal loss) and contribute to dysmyelinogenesis. Preclinical studies in animal models of the gangliosidoses have substantially improved lifespan and quality of life, leading to the initiation of several clinical trials. However, improvement of white matter pathology has lagged behind other metrics and few evidence-based explanations have been proposed to date. Research groups in the field are encouraged to include myelin-specific investigations in future gene therapy work to address this gap in knowledge.

Neuronal α-amylase is important for neuronal activity and glycogenolysis and reduces in presence of amyloid beta pathology.

Recent studies indicate a crucial role for neuronal glycogen storage and degradation in memory formation. We have previously identified alpha‐amylase (α‐amylase), a glycogen degradation enzyme, located within synaptic‐like structures in CA1 pyramidal neurons and shown that individuals with a high copy number variation of α‐amylase perform better on the episodic memory test. We reported that neuronal α‐amylase was absent in patients with Alzheimer's disease (AD) and that this loss corresponded to increased AD pathology. In the current study, we verified these findings in a larger patient cohort and determined a similar reduction in α‐amylase immunoreactivity in the molecular layer of hippocampus in AD patients. Next, we demonstrated reduced α‐amylase concentrations in oligomer amyloid beta 42 (Aβ42) stimulated SH‐SY5Y cells and neurons derived from human‐induced pluripotent stem cells (hiPSC) with PSEN1 mutation. Reduction of α‐amylase production and activity, induced by siRNA and α‐amylase inhibitor Tendamistat, respectively, was further shown to enhance glycogen load in SH‐SY5Y cells. Both oligomer Aβ42 stimulated SH‐SY5Y cells and hiPSC neurons with PSEN1 mutation showed, however, reduced load of glycogen. Finally, we demonstrate the presence of α‐amylase within synapses of isolated primary neurons and show that inhibition of α‐amylase activity with Tendamistat alters neuronal activity measured by calcium imaging. In view of these findings, we hypothesize that α‐amylase has a glycogen degrading function within synapses, potentially important in memory formation. Hence, a loss of α‐amylase, which can be induced by Aβ pathology, may in part underlie the disrupted memory formation seen in AD patients.

Neuronal storage disease in an adult female rabbit

Neuronal storage disease in an adult female rabbit

A Recurrent Gain-of-Function Mutation in CLCN6, Encoding the ClC-6 Cl−/H+-Exchanger, Causes Early-Onset Neurodegeneration

Dysfunction of the endolysosomal system is often associated with neurodegenerative disease because postmitotic neurons are particularly reliant on the elimination of intracellular aggregates. Adequate function of endosomes and lysosomes requires finely tuned luminal ion homeostasis and transmembrane ion fluxes. Endolysosomal CLC Cl-/H+ exchangers function as electric shunts for proton pumping and in luminal Cl- accumulation. We now report three unrelated children with severe neurodegenerative disease, who carry the same de novo c.1658A>G (p.Tyr553Cys) mutation in CLCN6, encoding the late endosomal Cl-/H+-exchanger ClC-6. Whereas Clcn6-/- mice have only mild neuronal lysosomal storage abnormalities, the affected individuals displayed severe developmental delay with pronounced generalized hypotonia, respiratory insufficiency, and variable neurodegeneration and diffusion restriction in cerebral peduncles, midbrain, and/or brainstem in MRI scans. The p.Tyr553Cys amino acid substitution strongly slowed ClC-6 gating and increased current amplitudes, particularly at the acidic pH of late endosomes. Transfection of ClC-6Tyr553Cys, but not ClC-6WT, generated giant LAMP1-positive vacuoles that were poorly acidified. Their generation strictly required ClC-6 ion transport, as shown by transport-deficient double mutants, and depended on Cl-/H+ exchange, as revealed by combination with the uncoupling p.Glu200Ala substitution. Transfection of either ClC-6Tyr553Cys/Glu200Ala or ClC-6Glu200Ala generated slightly enlarged vesicles, suggesting that p.Glu200Ala, previously associated with infantile spasms and microcephaly, is also pathogenic. Bafilomycin treatment abrogated vacuole generation, indicating that H+-driven Cl- accumulation osmotically drives vesicle enlargement. Our work establishes mutations in CLCN6 associated with neurological diseases, whose spectrum of clinical features depends on the differential impact of the allele on ClC-6 function.

A Single Injection of an Optimized Adeno-Associated Viral Vector into Cerebrospinal Fluid Corrects Neurological Disease in a Murine Model of GM1 Gangliosidosis.

GM1 gangliosidosis is a rare neurodegenerative lysosomal storage disease caused by loss-of-function mutations in the gene encoding beta-galactosidase (β-gal). There are no approved treatments for GM1 gangliosidosis. Previous studies in animal models have demonstrated that adeno-associated viral (AAV) vector-mediated gene transfer to the brain can restore β-gal expression and prevent the onset of neurological signs. We developed an optimized AAV vector expressing human β-gal and evaluated the efficacy of a single intracerebroventricular injection of this vector into the cerebrospinal fluid (CSF) of a murine disease model. The AAV vector administration into the CSF increased β-gal activity in the brain, reduced neuronal lysosomal storage lesions, prevented the onset of neurological signs and gait abnormalities, and increased survival. These findings demonstrate the potential therapeutic activity of this vector and support its subsequent development for the treatment of GM1 gangliosidosis.

Therapeutic benefit after intracranial gene therapy delivered during the symptomatic stage in a feline model of Sandhoff disease.

Sandhoff disease (SD) is an autosomal recessive lysosomal storage disease caused by defects in the β-subunit of β-N-acetylhexosaminidase (Hex), the enzyme that catabolizes GM2 ganglioside (GM2). Hex deficiency causes neuronal storage of GM2 and related glycoconjugates, resulting in progressive neurodegeneration and death, typically in infancy. No effective treatment exists for human patients. Adeno-associated virus (AAV) gene therapy led to improved clinical outcome and survival of SD cats treated before the onset of disease symptoms. Most human patients are diagnosed after clinical disease onset, so it is imperative to test AAV gene therapy in symptomatic SD cats to provide a realistic indication of therapeutic benefits that can be expected in humans. In this study, AAVrh8 vectors injected into the thalamus and deep cerebellar nuclei of symptomatic SD cats resulted in widespread central nervous system enzyme distribution, although a substantial burden of storage material remained. Cats treated in the early symptomatic phase showed delayed disease progression and a significant survival increase versus untreated cats. Treatment was less effective when administered later in the disease course, although therapeutic benefit was still possible. Results are encouraging for the treatment of human patients and provide support for the development AAV gene therapy for human SD.

Pharmacokinetics and distribution of 2-hydroxypropyl-β-cyclodextrin following a single intrathecal dose to cats.

2‐Hydroxypropyl‐β‐cyclodextrin (HP‐β‐CD) is an experimental therapy for Niemann‐Pick disease type C (NPC) that reduced neuronal cholesterol and ganglioside storage, reduced Purkinje cell death, and increased lifespan in npc1−/− mice and NPC1 cats. In this study, tissue distribution was investigated in normal cats that received a single 120‐mg dose of [14C]‐HP‐β‐CD (approximately 200 μCi/cat) via the cerebellomedullary cistern (CBMC) and lumbar cistern. One cat was euthanized at each of various time points up to 24 hours postdose for subsequent processing and quantitative whole‐body autoradiographic analysis. HP‐β‐CD‐derived radioactivity absorbed from the CBMC was widely distributed to cat tissues; most tissues were observed to have reached their highest concentration at 1 hour postdose. HP‐β‐CD‐derived radioactivity penetrated into the deeper parts of the central nervous system with the highest concentration at 4 hours (403 μg Eq/g or 0.28 mM) and remained high (49.7 μg Eq/g or 0.03 mM) at 24 hours. The relatively long half‐life (11‐30 hours) in cerebral ventricles and the subarachnoid space surrounding the brain and spinal cord might contribute to the efficacy of HP‐β‐CD in NPC1 cats. Other tissues with high concentrations of radioactivity were nasal turbinates, pituitary gland, and urinary bladder, while relatively low concentrations were observed in blood and bile.

Synapse alterations precede neuronal damage and storage pathology in a human cerebral organoid model of CLN3-juvenile neuronal ceroid lipofuscinosis

The juvenile form of neuronal ceroid Lipofuscinosis (JNCL) is the most common form within this group of rare lysosomal storage disorders, causing pediatric neurodegeneration. The genetic disorder, which is caused by recessive mutations affecting the CLN3 gene, features progressive vision loss, cognitive and motor decline and other psychiatric conditions, seizure episodes, leading to premature death. Animal models have traditionally aid the understanding of the disease mechanisms and pathology and are very relevant for biomarker research and therapeutic testing. Nevertheless, there is a need for establishing reliable and predictive human cellular models to study the disease. Since patient material, particularly from children, is scarce and difficult to obtain, we generated an engineered a CLN3-mutant isogenic human induced pluripotent stem cell (hiPSC) line carrying the c.1054C → T pathologic variant, using state of the art CRISPR/Cas9 technology. To prove the suitability of the isogenic pair to model JNCL, we screened for disease-specific phenotypes in non-neuronal two-dimensional cell culture models as well as in cerebral brain organoids. Our data demonstrates that the sole introduction of the pathogenic variant gives rise to classical hallmarks of JNCL in vitro. Additionally, we discovered an alteration of the splicing caused by this particular mutation. Next, we derived cerebral organoids and used them as a neurodevelopmental model to study the particular effects of the CLN3Q352X mutation during brain formation in the disease context. About half of the mutation -carrying cerebral organoids completely failed to develop normally. The other half, which escaped this severe defect were used for the analysis of more subtle alterations. In these escapers, whole-transcriptome analysis demonstrated early disease signatures, affecting pathways related to development, corticogenesis and synapses. Complementary metabolomics analysis confirmed decreased levels of cerebral tissue metabolites, some particularly relevant for synapse formation and neurotransmission, such as gamma-amino butyric acid (GABA). Our data suggests that a mutation in CLN3 severely affects brain development. Furthermore, before disease onset, disease -associated neurodevelopmental changes, particular concerning synapse formation and function, occur.