Epileptic Encephalopathy Caused by Mutations in the Guanine Nucleotide Exchange Factor DENND5A.

Epileptic encephalopathies are a catastrophic group of epilepsies characterized by refractory seizures and cognitive arrest, often resulting from abnormal brain development. Here, we have identified an epileptic encephalopathy additionally featuring cerebral calcifications and coarse facial features caused by recessive loss-of-function mutations in DENND5A. DENND5A contains a DENN domain, an evolutionarily ancient enzymatic module conferring guanine nucleotide exchange factor (GEF) activity to multiple proteins serving as GEFs for Rabs, which are key regulators of membrane trafficking. DENND5A is detected predominantly in neuronal tissues, and its highest levels occur during development. Knockdown of DENND5A leads to striking alterations in neuronal development. Mechanistically, these changes appear to result from upregulation of neurotrophin receptors, leading to enhanced downstream signaling. Thus, we have identified a link between a DENN domain protein and neuronal development, dysfunction of which is responsible for a form of epileptic encephalopathy.

Formaldehyde induces diabetes-associated cognitive impairments.

Patients with type 2 diabetes mellitus (T2DM) often develop cognitive impairments and have an increased risk of developing Alzheimer's disease. Hyperglycemia is a major characteristic of T2DM, but how elevated glucose levels lead to cognitive decline remains elusive. Here, we report that patients with T2DM and mutations in the formaldehyde (FA)-degrading enzyme aldehyde dehydrogenase 2 ( ALDH2) gene had higher levels of FA and more severe dementia. Injection of FA induced hyperglycemia and cognitive deficits in rats. Ablation of gene expression of ALDH2, the main enzyme to oxidize FA, resulted in abnormally high levels of hippocampal FA, leading to hyperglycemia and cognitive impairments as well as potentiating streptozotocin-induced diabetes development in ALDH2 knockout mice. We found that FA interacts with insulin to form FA-insulin adducts, and these FA-insulin adducts caused insulin deficiency, contributing to memory decline in diabetic rodent models. Reduction of FA by transgenic overexpression of human ALDH2 attenuates hyperglycemia and alleviates cognitive deficits in diabetic mouse models. These findings suggest that excess FA plays a critical role in mediating diabetes-related dementia. Targeting FA and its metabolizing enzyme ALDH2 may be a valid approach for preventing and treating dementia in diabetes mellitus.-Tan, T., Zhang, Y., Luo, W., Lv, J., Han, C., Hamlin, J. N. R., Luo, H., Li, H., Wan, Y., Yang, X., Song, W., Tong, Z. Formaldehyde induces diabetes-associated cognitive impairments.

Intersectin-s interaction with DENND2B facilitates recycling of epidermal growth factor receptor.

Epidermal growth factor (EGF) activates the EGF receptor (EGFR) and stimulates its internalization and trafficking to lysosomes for degradation. However, a percentage of EGFR undergoes ligand-independent endocytosis and is rapidly recycled back to the plasma membrane. Importantly, alterations in EGFR recycling are a common hallmark of cancer, and yet, our understanding of the machineries controlling the fate of endocytosed EGFR is incomplete. Intersectin-s is a multi-domain adaptor protein that is required for internalization of EGFR Here, we discover that intersectin-s binds DENND2B, a guanine nucleotide exchange factor for the exocytic GTPase Rab13, and this interaction promotes recycling of ligand-free EGFR to the cell surface. Intriguingly, upon EGF treatment, DENND2B is phosphorylated by protein kinase D and dissociates from intersectin-s, allowing for receptor targeting to degradation. Our study thus reveals a novel mechanism controlling the fate of internalized EGFR with important implications for cancer.

Modeling Parkinson's disease pathology in human dopaminergic neurons by sequential exposure to α-synuclein fibrils and proinflammatory cytokines.

Lewy bodies (LBs), α-synuclein-enriched intracellular inclusions, are a hallmark of Parkinson's disease (PD) pathology, yet a cellular model for LB formation remains elusive. Recent evidence indicates that immune dysfunction may contribute to the development of PD. In this study, we found that induced pluripotent stem cell (iPSC)-derived human dopaminergic (DA) neurons form LB-like inclusions after treatment with α-synuclein preformed fibrils (PFFs) but only when coupled to a model of immune challenge (interferon-γ or interleukin-1ß treatment) or when co-cultured with activated microglia-like cells. Exposure to interferon-γ impairs lysosome function in DA neurons, contributing to LB formation. The knockdown of LAMP2 or the knockout of GBA in conjunction with PFF administration is sufficient for inclusion formation. Finally, we observed that the LB-like inclusions in iPSC-derived DA neurons are membrane bound, suggesting that they are not limited to the cytoplasmic compartment but may be formed due to dysfunctions in autophagy. Together, these data indicate that immune-triggered lysosomal dysfunction may contribute to the development of PD pathology.

Loss of symmetric cell division of apical neural progenitors drives DENND5A-related developmental and epileptic encephalopathy.

Developmental and epileptic encephalopathies (DEEs) feature altered brain development, developmental delay and seizures, with seizures exacerbating developmental delay. Here we identify a cohort with biallelic variants in DENND5A, encoding a membrane trafficking protein, and develop animal models with phenotypes like the human syndrome. We demonstrate that DENND5A interacts with Pals1/MUPP1, components of the Crumbs apical polarity complex required for symmetrical division of neural progenitor cells. Human induced pluripotent stem cells lacking DENND5A fail to undergo symmetric cell division with an inherent propensity to differentiate into neurons. These phenotypes result from misalignment of the mitotic spindle in apical neural progenitors. Cells lacking DENND5A orient away from the proliferative apical domain surrounding the ventricles, biasing daughter cells towards a more fate-committed state, ultimately shortening the period of neurogenesis. This study provides a mechanism for DENND5A-related DEE that may be generalizable to other developmental conditions and provides variant-specific clinical information for physicians and families.

Loss of symmetric cell division of apical neural progenitors drives DENND5A-related developmental and epileptic encephalopathy.

Developmental and epileptic encephalopathies (DEEs) are a heterogenous group of epilepsies in which altered brain development leads to developmental delay and seizures, with the epileptic activity further negatively impacting neurodevelopment. Identifying the underlying cause of DEEs is essential for progress toward precision therapies. Here we describe a group of individuals with biallelic variants in DENND5A and determine that variant type is correlated with disease severity. We demonstrate that DENND5A interacts with MUPP1 and PALS1, components of the Crumbs apical polarity complex, which is required for both neural progenitor cell identity and the ability of these stem cells to divide symmetrically. Induced pluripotent stem cells lacking DENND5A fail to undergo symmetric cell division during neural induction and have an inherent propensity to differentiate into neurons, and transgenic DENND5A mice, with phenotypes like the human syndrome, have an increased number of neurons in the adult subventricular zone. Disruption of symmetric cell division following loss of DENND5A results from misalignment of the mitotic spindle in apical neural progenitors. A subset of DENND5A is localized to centrosomes, which define the spindle poles during mitosis. Cells lacking DENND5A orient away from the proliferative apical domain surrounding the ventricles, biasing daughter cells towards a more fate-committed state and ultimately shortening the period of neurogenesis. This study provides a mechanism behind DENND5A-related DEE that may be generalizable to other developmental conditions and provides variant-specific clinical information for physicians and families.

Rapid macropinocytic transfer of α-synuclein to lysosomes.

The nervous system spread of alpha-synuclein fibrils is thought to cause Parkinson's disease (PD) and other synucleinopathies; however, the mechanisms underlying internalization and cellular spread are enigmatic. Here, we use confocal and superresolution microscopy, subcellular fractionation, and electron microscopy (EM) of immunogold-labeled α-synuclein preformed fibrils (PFFs) to demonstrate that this form of the protein undergoes rapid internalization and is targeted directly to lysosomes in as little as 2 min. Uptake of PFFs is disrupted by macropinocytic inhibitors and circumvents classical endosomal pathways. Immunogold-labeled PFFs are seen at the highly curved inward edge of membrane ruffles, in newly formed macropinosomes, in multivesicular bodies and in lysosomes. While most fibrils remain in lysosomes, a portion is transferred to neighboring naive cells along with markers of exosomes. These data indicate that PFFs use a unique internalization mechanism as a component of cell-to-cell propagation.

Early defects in mucopolysaccharidosis type IIIC disrupt excitatory synaptic transmission.

The majority of patients affected with lysosomal storage disorders (LSD) exhibit neurological symptoms. For mucopolysaccharidosis type IIIC (MPSIIIC), the major burdens are progressive and severe neuropsychiatric problems and dementia, primarily thought to stem from neurodegeneration. Using the MPSIIIC mouse model, we studied whether clinical manifestations preceding massive neurodegeneration arise from synaptic dysfunction. Reduced levels or abnormal distribution of multiple synaptic proteins were revealed in cultured hippocampal and CA1 pyramidal MPSIIIC neurons. These defects were rescued by virus-mediated gene correction. Dendritic spines were reduced in pyramidal neurons of mouse models of MPSIIIC and other (Tay-Sachs, sialidosis) LSD as early as at P10. MPSIIIC neurons also presented alterations in frequency and amplitude of miniature excitatory and inhibitory postsynaptic currents, sparse synaptic vesicles, reduced postsynaptic densities, disorganized microtubule networks, and partially impaired axonal transport of synaptic proteins. Furthermore, postsynaptic densities were reduced in postmortem cortices of human MPS patients, suggesting that the pathology is a common hallmark for neurological LSD. Together, our results demonstrate that lysosomal storage defects cause early alterations in synaptic structure and abnormalities in neurotransmission originating from impaired synaptic vesicular transport, and they suggest that synaptic defects could be targeted to treat behavioral and cognitive defects in neurological LSD patients.

Nanotherapeutic Modulation of Human Neural Cells and Glioblastoma in Organoids and Monocultures.

Inflammatory processes in the brain are orchestrated by microglia and astrocytes in response to activators such as pathogen-associated molecular patterns, danger-associated molecular patterns and some nanostructures. Microglia are the primary immune responders in the brain and initiate responses amplified by astrocytes through intercellular signaling. Intercellular communication between neural cells can be studied in cerebral organoids, co-cultures or in vivo. We used human cerebral organoids and glioblastoma co-cultures to study glia modulation by dendritic polyglycerol sulfate (dPGS). dPGS is an extensively studied nanostructure with inherent anti-inflammatory properties. Under inflammatory conditions, lipocalin-2 levels in astrocytes are markedly increased and indirectly enhanced by soluble factors released from hyperactive microglia. dPGS is an effective anti-inflammatory modulator of these markers. Our results show that dPGS can enter neural cells in cerebral organoids and glial cells in monocultures in a time-dependent manner. dPGS markedly reduces lipocalin-2 abundance in the neural cells. Glioblastoma tumoroids of astrocytic origin respond to activated microglia with enhanced invasiveness, whereas conditioned media from dPGS-treated microglia reduce tumoroid invasiveness. Considering that many nanostructures have only been tested in cancer cells and rodent models, experiments in human 3D cerebral organoids and co-cultures are complementary in vitro models to evaluate nanotherapeutics in the pre-clinical setting. Thoroughly characterized organoids and standardized procedures for their preparation are prerequisites to gain information of translational value in nanomedicine. This study provides data for a well-characterized dendrimer (dPGS) that modulates the activation state of human microglia implicated in brain tumor invasiveness.

Open Science Meets Stem Cells: A New Drug Discovery Approach for Neurodegenerative Disorders.

Neurodegenerative diseases are a challenge for drug discovery, as the biological mechanisms are complex and poorly understood, with a paucity of models that faithfully recapitulate these disorders. Recent advances in stem cell technology have provided a paradigm shift, providing researchers with tools to generate human induced pluripotent stem cells (iPSCs) from patient cells. With the potential to generate any human cell type, we can now generate human neurons and develop "first-of-their-kind" disease-relevant assays for small molecule screening. Now that the tools are in place, it is imperative that we accelerate discoveries from the bench to the clinic. Using traditional closed-door research systems raises barriers to discovery, by restricting access to cells, data and other research findings. Thus, a new strategy is required, and the Montreal Neurological Institute (MNI) and its partners are piloting an "Open Science" model. One signature initiative will be that the MNI biorepository will curate and disseminate patient samples in a more accessible manner through open transfer agreements. This feeds into the MNI open drug discovery platform, focused on developing industry-standard assays with iPSC-derived neurons. All cell lines, reagents and assay findings developed in this open fashion will be made available to academia and industry. By removing the obstacles many universities and companies face in distributing patient samples and assay results, our goal is to accelerate translational medical research and the development of new therapies for devastating neurodegenerative disorders.

Ribosylation triggering Alzheimer's disease-like Tau hyperphosphorylation via activation of CaMKII.

Type 2 diabetes mellitus (T2DM) is regarded as one of the serious risk factors for age-related cognitive impairment; however, a causal link between these two diseases has so far not been established. It was recently discovered that, apart from high D-glucose levels, T2DM patients also display abnormally high concentrations of uric D-ribose. Here, we show for the first time that the administration of D-ribose, the most active glycator among monosaccharides, produces high levels of advanced glycation end products (AGEs) and, importantly, triggers hyperphosphorylation of Tau in the brain of C57BL/6 mouse and neuroblastoma N2a cells. However, the administration of D-glucose showed no significant changes in Tau phosphorylation under the same experimental conditions. Crucially, suppression of AGE formation using an AGEs inhibitor (aminoguanidine) effectively prevents hyperphosphorylation of Tau protein. Further study shows AGEs resulted from ribosylation activate calcium-/calmodulin-dependent protein kinase type II (CaMKII), a key kinase responsible for Tau hyperphosphorylation. These data suggest that there is indeed a mechanistic link between ribosylation and Tau hyperphosphorylation. Targeting ribosylation by inhibiting AGE formation may be a promising therapeutic strategy to prevent Alzheimer's disease-like Tau hyperphosphorylation and diabetic encephalopathies.

Age-related formaldehyde interferes with DNA methyltransferase function, causing memory loss in Alzheimer's disease.

Hippocampus-related topographic amnesia is the most common symptom of memory disorders in Alzheimer's disease (AD) patients. Recent studies have revealed that experience-mediated DNA methylation, which is regulated by enzymes with DNA methyltransferase (DNMT) activity, is required for the formation of recent memory as well as the maintenance of remote memory. Notably, overexpression of DNMT3a in the hippocampus can reverse spatial memory deficits in aged mice. However, a decline in global DNA methylation was found in the autopsied hippocampi of patients with AD. Exactly, what endogenous factors that affect DNA methylation still remain to be elucidated. Here, we report a marked increase in endogenous formaldehyde levels is associated with a decline in global DNA methylation in the autopsied hippocampus from AD patients. In vitro and in vivo results show that formaldehyde in excess of normal physiological levels reduced global DNA methylation by interfering DNMTs. Interestingly, intrahippocampal injection of excess formaldehyde before spatial learning in healthy adult rats can mimic the learning difficulty of early stage of AD. Moreover, injection of excess formaldehyde after spatial learning can mimic the loss of remote spatial memory observed in late stage of AD. These findings suggest that aging-associated formaldehyde contributes to topographic amnesia in AD patients.

Accumulated hippocampal formaldehyde induces age-dependent memory decline.

Aging is an important factor in memory decline in aged animals and humans and in Alzheimer's disease and is associated with the impairment of hippocampal long-term potentiation (LTP) and down-regulation of NR1/NR2B expression. Gaseous formaldehyde exposure is known to induce animal memory loss and human cognitive decline; however, it is unclear whether the concentrations of endogenous formaldehyde are elevated in the hippocampus and how excess formaldehyde affects LTP and memory formation during the aging process. In the present study, we report that hippocampal formaldehyde accumulated in memory-deteriorating diseases such as age-related dementia. Spatial memory performance was gradually impaired in normal Sprague-Dawley rats by persistent intraperitoneal injection with formaldehyde. Furthermore, excess formaldehyde treatment suppressed the hippocampal LTP formation by blocking N-methyl-D-aspartate (NMDA) receptor. Chronic excess formaldehyde treatment over a period of 30 days markedly decreased the viability of the hippocampus and down-regulated the expression of the NR1 and NR2B subunits of the NMDA receptor. Our results indicate that excess endogenous formaldehyde is a critical factor in memory loss in age-related memory-deteriorating diseases.

Aging-associated excess formaldehyde leads to spatial memory deficits.

Recent studies show that formaldehyde participates in DNA demethylation/methylation cycle. Emerging evidence identifies that neuronal activity induces global DNA demethylation and re-methylation; and DNA methylation is a critical step for memory formation. These data suggest that endogenous formaldehyde may intrinsically link learning-responsive DNA methylation status and memory formation. Here, we report that during spatial memory formation process, spatial training induces an initial global DNA demethylation and subsequent re-methylation associated with hippocampal formaldehyde elevation then decline to baseline level in Sprague Dawley rats. Scavenging this elevated formaldehyde by formaldehyde-degrading enzyme (FDH), or enhancing DNA demethylation by a DNA demethylating agent, both led to spatial memory deficits by blocking DNA re-methylation in rats. Furthermore, we found that the normal adult rats intrahippocampally injected with excess formaldehyde can imitate the aged-related spatial memory deficits and global DNA methylation decline. These findings indicate that aging-associated excess formaldheyde contributes to cognitive decline during aging.

D-ribose induces cellular protein glycation and impairs mouse spatial cognition.

BACKGROUND: D-ribose, an important reducing monosaccharide, is highly active in the glycation of proteins, and results in the rapid production of advanced glycation end products (AGEs) in vitro. However, whether D-ribose participates in glycation and leads to production of AGEs in vivo still requires investigation. METHODOLOGY/PRINCIPAL FINDINGS: Here we treated cultured cells and mice with D-ribose and D-glucose to compare ribosylation and glucosylation for production of AGEs. Treatment with D-ribose decreased cell viability and induced more AGE accumulation in cells. C57BL/6J mice intraperitoneally injected with D-ribose for 30 days showed high blood levels of glycated proteins and AGEs. Administration of high doses D-ribose also accelerated AGE formation in the mouse brain and induced impairment of spatial learning and memory ability according to the performance in Morris water maze test. CONCLUSIONS/SIGNIFICANCE: These data demonstrate that D-ribose but not D-glucose reacts rapidly with proteins and produces significant amounts of AGEs in both cultured cells and the mouse brain, leading to accumulation of AGEs which may impair mouse spatial cognition.