Alán Alpár

Molecular reorganization of endocannabinoid signalling in Alzheimer’s disease

Retrograde messengers adjust the precise timing of neurotransmitter release from the presynapse, thus modulating synaptic efficacy and neuronal activity. 2-Arachidonoyl glycerol, an endocannabinoid, is one such messenger produced in the postsynapse that inhibits neurotransmitter release upon activating presynaptic CB(1) cannabinoid receptors. Cognitive decline in Alzheimers disease is due to synaptic failure in hippocampal neuronal networks. We hypothesized that errant retrograde 2-arachidonoyl glycerol signalling impairs synaptic neurotransmission in Alzheimers disease. Comparative protein profiling and quantitative morphometry showed that overall CB(1) cannabinoid receptor protein levels in the hippocampi of patients with Alzheimers disease remain unchanged relative to age-matched controls, and CB(1) cannabinoid receptor-positive presynapses engulf amyloid-β-containing senile plaques. Hippocampal protein concentrations for the sn-1-diacylglycerol lipase α and β isoforms, synthesizing 2-arachidonoyl glycerol, significantly increased in definite Alzheimers (Braak stage VI), with ectopic sn-1-diacylglycerol lipase β expression found in microglia accumulating near senile plaques and apposing CB(1) cannabinoid receptor-positive presynapses. We found that microglia, expressing two 2-arachidonoyl glycerol-degrading enzymes, serine hydrolase α/β-hydrolase domain-containing 6 and monoacylglycerol lipase, begin to surround senile plaques in probable Alzheimers disease (Braak stage III). However, Alzheimers pathology differentially impacts serine hydrolase α/β-hydrolase domain-containing 6 and monoacylglycerol lipase in hippocampal neurons: serine hydrolase α/β-hydrolase domain-containing 6 expression ceases in neurofibrillary tangle-bearing pyramidal cells. In contrast, pyramidal cells containing hyperphosphorylated tau retain monoacylglycerol lipase expression, although at levels significantly lower than in neurons lacking neurofibrillary pathology. Here, monoacylglycerol lipase accumulates in CB(1) cannabinoid receptor-positive presynapses. Subcellular fractionation revealed impaired monoacylglycerol lipase recruitment to biological membranes in post-mortem Alzheimers tissues, suggesting that disease progression slows the termination of 2-arachidonoyl glycerol signalling. We have experimentally confirmed that altered 2-arachidonoyl glycerol signalling could contribute to synapse silencing in Alzheimers disease by demonstrating significantly prolonged depolarization-induced suppression of inhibition when superfusing mouse hippocampi with amyloid-β. We propose that the temporal dynamics and cellular specificity of molecular rearrangements impairing 2-arachidonoyl glycerol availability and actions may differ from those of anandamide. Thus, enhanced endocannabinoid signalling, particularly around senile plaques, can exacerbate synaptic failure in Alzheimers disease.

Miswiring the brain: Δ9‐tetrahydrocannabinol disrupts cortical development by inducing an SCG10/stathmin‐2 degradation pathway

Children exposed in utero to cannabis present permanent neurobehavioral and cognitive impairments. Psychoactive constituents from Cannabis spp., particularly Δ9‐tetrahydrocannabinol (THC), bind to cannabinoid receptors in the fetal brain. However, it is unknown whether THC can trigger a cannabinoid receptor‐driven molecular cascade to disrupt neuronal specification. Here, we show that repeated THC exposure disrupts endocannabinoid signaling, particularly the temporal dynamics of CB1 cannabinoid receptor, to rewire the fetal cortical circuitry. By interrogating the THC‐sensitive neuronal proteome we identify Superior Cervical Ganglion 10 (SCG10)/stathmin‐2, a microtubule‐binding protein in axons, as a substrate of altered neuronal connectivity. We find SCG10 mRNA and protein reduced in the hippocampus of midgestational human cannabis‐exposed fetuses, defining SCG10 as the first cannabis‐driven molecular effector in the developing cerebrum. CB1 cannabinoid receptor activation recruits c‐Jun N‐terminal kinases to phosphorylate SCG10, promoting its rapid degradation in situ in motile axons and microtubule stabilization. Thus, THC enables ectopic formation of filopodia and alters axon morphology. These data highlight the maintenance of cytoskeletal dynamics as a molecular target for cannabis, whose imbalance can limit the computational power of neuronal circuitries in affected offspring.

Molecular interrogation of hypothalamic organization reveals distinct dopamine neuronal subtypes

The hypothalamus contains the highest diversity of neurons in the brain. Many of these neurons can co-release neurotransmitters and neuropeptides in a use-dependent manner. Investigators have hitherto relied on candidate protein-based tools to correlate behavioral, endocrine and gender traits with hypothalamic neuron identity. Here we map neuronal identities in the hypothalamus by single-cell RNA sequencing. We distinguished 62 neuronal subtypes producing glutamatergic, dopaminergic or GABAergic markers for synaptic neurotransmission and harboring the ability to engage in task-dependent neurotransmitter switching. We identified dopamine neurons that uniquely coexpress the Onecut3 and Nmur2 genes, and placed these in the periventricular nucleus with many synaptic afferents arising from neuromedin S+ neurons of the suprachiasmatic nucleus. These neuroendocrine dopamine cells may contribute to the dopaminergic inhibition of prolactin secretion diurnally, as their neuromedin S+ inputs originate from neurons expressing Per2 and Per3 and their tyrosine hydroxylase phosphorylation is regulated in a circadian fashion. Overall, our catalog of neuronal subclasses provides new understanding of hypothalamic organization and function.

The carbonyl scavengers aminoguanidine and tenilsetam protect against the neurotoxic effects of methylglyoxal

Advanced glycation end products (AGEs) have been identified in age-related intracellular protein deposits of Alzheimer’s disease (amyloid plaques and neurofibrillary tangles) and Parkinson disease (Lewy bodies), suggesting that these protein deposits have been exposed to AGE precursors such as the reactive dicarbonyl compound methylglyoxal. In ageing tissue and under diabetic pseudohypoxia, intracellular methylglyoxal levels rise through an impairment of triosephosphate utilization. Furthermore, methylglyoxal detoxification is impaired when reduced glutathione levels are low, conditions, which have all been described in Alzheimer’s disease. However, there is less known about the toxicity of methylglyoxal, particularly about therapeutic strategies to scavenge such dicarbonyl compounds and attenuate their toxicity. In our study, extracellularly applied methylglyoxal was shown to be toxic to human neuroblastoma cells in a dose-dependent manner above concentrations of 150 µM with a LD50 of approximately 1.25 mM. Pre-incubation of methylglyoxal with a variety of carbonyl scavengers such as aminoguanidine or tenilsetam and the thiol antioxidant lipoic acid significantly reduced its toxicity. In summary, carbonyl scavengers might offer a promising therapeutic strategy to reduce the neurotoxicity of reactive carbonyl compounds, providing a potential benefit for patients with age-related neurodegenerative diseases.

Different dendrite and dendritic spine alterations in basal and apical arbors in mutant human amyloid precursor protein transgenic mice.

The extracellular deposition of amyloid-beta peptide (Abeta) in brain parenchyma is one of the characteristic features of Alzheimers disease and is suggested to induce reactive and degenerative changes in neuronal cell bodies, axons and dendritic processes. In particular, within and in close proximity to amyloid plaques, distinctive morphological alterations have been observed, including changes in neurite trajectory and decreases in dendritic diameter and in spine density. Apart from these plaque-associated focal aberrations, little is known regarding modifications of the global dendritic morphology including the detailed and comparative quantitative analysis of apical and basal arbors. The objective of the present study was to investigate the effects of amyloid plaque deposition and elevated soluble Abeta on neuronal morphology in mutant human amyloid precursor protein (hAPP) transgenic mice (line Tg2576; [K. Hsiao, P. Chapman, S. Nilsen, C. Eckman, Y. Harigaya, S. Younkin, F. Yang, G. Cole, Correlative memory deficits, Abeta elevation, and amyloid plaques in transgenic mice, Science 274 (1996) 99-102]). Retrogradelly labeled callosal-projecting pyramidal cells in the primary somatosensory cortex were three-dimensionally analyzed. Although basal dendrites remained unaffected, analysis of apical trees revealed a number of unambiguous morphological changes. Thus, in TG2576 mice, the apical arbors were shortened in total length and less branched. Furthermore, the diameter of proximal dendritic segments was increased whereas that of distal segments was reduced. Analysis of spine numbers and distribution on basal and apical trees demonstrated a significant reduction in spine densities along the whole course of dendrites. The findings suggest that Abeta-related pathology induces morphological aberrations in basal and apical arbors to different degrees which are unrelated to direct plaque-associated changes.

Distribution of pyramidal cells associated with perineuronal nets in the neocortex of rat

Perineuronal nets are lattice-like accumulations of extracellular matrix components around the cell body and perisomatic portion of certain neurons. Whereas interneurons associated to this specific neuron-associated sheath have been elaborately classified, less effort has been undertaken to describe the occurrence of perineuronal nets around pyramidal neurons. Our aim was to give a detailed and comparative description of the occurrence of net-associated pyramidal cells throughout the rat neocortex as well as to systematically and comparatively analyze the relation of main projection types of principal neurons to the presence of perineuronal nets. The present study revealed that perineuronal nets stained with WFA were associated rather rarely to pyramidal cells compared to interneurons in layers II/III and V/VI of rat neocortex. However, their frequency was considerably different between various cortical areas with a maximum in visual cortex and with a minimum in secondary motor cortices. Further analysis revealed that neuron-associated matrix sheaths around principal cells were more common in the primary than in the secondary fields of corresponding areas and they were more numerous in infra-than in supragranular layers in most regions. Subfields of cortical areas also differed regarding the occurrence of net-associated principal cells, and the subtlety of cortical representation seemed to correlate with the frequency of perineuronal nets around pyramidal neurons in the primary somatosensory cortex. It appears that net-associated pyramidal cells do not have a projection pattern restricted to distinct target regions. Rather a functional heterogeneity of the pyramidal cell population contributing to specific intra-or subcortical projections is suggested.

Neurochemical mapping of the human hippocampus reveals perisynaptic matrix around functional synapses in Alzheimer’s disease

Perineuronal matrix is an extracellular protein scaffold to shape neuronal responsiveness and survival. Whilst perineuronal nets engulf the somatodendritic axis of neurons, axonal coats are focal extracellular protein aggregates surrounding individual synapses. Here, we addressed the chemical identity and subcellular localization of both perineuronal and perisynaptic matrices in the human hippocampus, whose neuronal circuitry is progressively compromised in Alzheimer’s disease. We hypothesized that (1) the cellular expression sites of chondroitin sulphate proteoglycan-containing extracellular matrix associate with specific neuronal identities, reflecting network dynamics, and (2) the regional distribution and molecular composition of axonal coats must withstand Alzheimer’s disease-related modifications to protect functional synapses. We show by epitope-specific antibodies that the perineuronal protomap of the human hippocampus is distinct from other mammals since pyramidal cells but not calretinin+ and calbindin+ interneurons, neurochemically classified as novel neuronal subtypes, lack perineuronal nets. We find that cartilage link protein-1 and brevican-containing matrices form isolated perisynaptic coats, engulfing both inhibitory and excitatory terminals in the dentate gyrus and entorhinal cortex. Ultrastructural analysis revealed that presynaptic neurons contribute components of perisynaptic coats via axonal transport. We demonstrate, by combining biochemical profiling and neuroanatomy in Alzheimer’s patients and transgenic (APdE9) mice, the preserved turnover and distribution of axonal coats around functional synapses along dendrite segments containing hyperphosphorylated tau and in amyloid-β-laden hippocampal microdomains. We conclude that the presynapse-driven formation of axonal coats is a candidate mechanism to maintain synapse integrity under neurodegenerative conditions.

Dietary energy substrates reverse early neuronal hyperactivity in a mouse model of Alzheimer's disease.

Deficient energy metabolism and network hyperactivity are the early symptoms of Alzheimers disease (AD). In this study, we show that administration of exogenous oxidative energy substrates (OES) corrects neuronal energy supply deficiency that reduces the amyloid‐beta‐induced abnormal neuronal activity in vitro and the epileptic phenotype in AD model in vivo. In vitro, acute application of protofibrillar amyloid‐β1–42 (Aβ1–42) induced aberrant network activity in wild‐type hippocampal slices that was underlain by depolarization of both the neuronal resting membrane potential and GABA‐mediated current reversal potential. Aβ1–42 also impaired synaptic function and long‐term potentiation. These changes were paralleled by clear indications of impaired energy metabolism, as indicated by abnormal NAD(P)H signaling induced by network activity. However, when glucose was supplemented with OES pyruvate and 3‐beta‐hydroxybutyrate, Aβ1–42 failed to induce detrimental changes in any of the above parameters. We administered the same OES as chronic supplementation to a standard diet to APPswe/PS1dE9 transgenic mice displaying AD‐related epilepsy phenotype. In the ex‐vivo slices, we found neuronal subpopulations with significantly depolarized resting and GABA‐mediated current reversal potentials, mirroring abnormalities we observed under acute Aβ1‐42 application. Ex‐vivo cortex of transgenic mice fed with standard diet displayed signs of impaired energy metabolism, such as abnormal NAD(P)H signaling and strongly reduced tolerance to hypoglycemia. Transgenic mice also possessed brain glycogen levels twofold lower than those of wild‐type mice. However, none of the above neuronal and metabolic dysfunctions were observed in transgenic mice fed with the OES‐enriched diet. In vivo, dietary OES supplementation abated neuronal hyperexcitability, as the frequency of both epileptiform discharges and spikes was strongly decreased in the APPswe/PS1dE9 mice placed on the diet. Altogether, our results suggest that early AD‐related neuronal malfunctions underlying hyperexcitability and energy metabolism deficiency can be prevented by dietary supplementation with native energy substrates.

The renaissance of Ca2+-binding proteins in the nervous system: secretagogin takes center stage.

Effective control of the Ca(2+) homeostasis in any living cell is paramount to coordinate some of the most essential physiological processes, including cell division, morphological differentiation, and intercellular communication. Therefore, effective homeostatic mechanisms have evolved to maintain the intracellular Ca(2+) concentration at physiologically adequate levels, as well as to regulate the spatial and temporal dynamics of Ca(2+)signaling at subcellular resolution. Members of the superfamily of EF-hand Ca(2+)-binding proteins are effective to either attenuate intracellular Ca(2+) transients as stochiometric buffers or function as Ca(2+) sensors whose conformational change upon Ca(2+) binding triggers protein-protein interactions, leading to cell state-specific intracellular signaling events. In the central nervous system, some EF-hand Ca(2+)-binding proteins are restricted to specific subtypes of neurons or glia, with their expression under developmental and/or metabolic control. Therefore, Ca(2+)-binding proteins are widely used as molecular markers of cell identity whilst also predicting excitability and neurotransmitter release profiles in response to electrical stimuli. Secretagogin is a novel member of the group of EF-hand Ca(2+)-binding proteins whose expression precedes that of many other Ca(2+)-binding proteins in postmitotic, migratory neurons in the embryonic nervous system. Secretagogin expression persists during neurogenesis in the adult brain, yet becomes confined to regionalized subsets of differentiated neurons in the adult central and peripheral nervous and neuroendocrine systems. Secretagogin may be implicated in the control of neuronal turnover and differentiation, particularly since it is re-expressed in neoplastic brain and endocrine tumors and modulates cell proliferation in vitro. Alternatively, and since secretagogin can bind to SNARE proteins, it might function as a Ca(2+) sensor/coincidence detector modulating vesicular exocytosis of neurotransmitters, neuropeptides or hormones. Thus, secretagogin emerges as a functionally multifaceted Ca(2+)-binding protein whose molecular characterization can unravel a new and fundamental dimension of Ca(2+)signaling under physiological and disease conditions in the nervous system and beyond.

Nerve growth factor scales endocannabinoid signaling by regulating monoacylglycerol lipase turnover in developing cholinergic neurons.

Endocannabinoid, particularly 2-arachidonoyl glycerol (2-AG), signaling has recently emerged as a molecular determinant of neuronal migration and synapse formation during cortical development. However, the cell type specificity and molecular regulation of spatially and temporally confined morphogenic 2-AG signals remain unexplored. Here, we demonstrate that genetic and pharmacological manipulation of CB1 cannabinoid receptors permanently alters cholinergic projection neuron identity and hippocampal innervation. We show that nerve growth factor (NGF), implicated in the morphogenesis and survival of cholinergic projection neurons, dose-dependently and coordinately regulates the molecular machinery for 2-AG signaling via tropomyosine kinase A receptors in vitro. In doing so, NGF limits the sorting of monoacylglycerol lipase (MGL), rate limiting 2-AG bioavailability, to proximal neurites, allowing cell-autonomous 2-AG signaling at CB1 cannabinoid receptors to persist at atypical locations to induce superfluous neurite extension. We find that NGF controls MGL degradation in vitro and in vivo and identify the E3 ubiquitin ligase activity of breast cancer type 1 susceptibility protein (BRCA1) as a candidate facilitating MGL’s elimination from motile neurite segments, including growth cones. BRCA1 inactivation by cisplatin or genetically can rescue and reposition MGL, arresting NGF-induced growth responses. These data indicate that NGF can orchestrate endocannabinoid signaling to promote cholinergic differentiation and implicate BRCA1 in determining neuronal morphology.