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Background information The endo-lysosomal system (ELS) comprises a set of membranous organelles responsible for transporting intracellular and extracellular components within cells. Defects in lysosomal proteins usually affect a large variety of processes and underlie many diseases, most of them with a strong neuronal impact. Mutations in the endoplasmic reticulum-resident CLN8 protein cause CLN8 disease. This condition is one of the 14 known Neuronal Ceroid Lipofuscinoses (NCL), a group of inherited diseases characterized by accumulation of lipofuscin-like pigments within lysosomes. Besides mediating the transport of soluble lysosomal proteins, recent research suggested a role for CLN8 in the transport of vesicles and lipids, and autophagy. However, the consequences of CLN8 deficiency on ELS structure and activity, as well as the potential impact on neuronal development, remain poorly characterized. Therefore, we performed CLN8 knockdown in neuronal and non-neuronal cell models to analyze structural, dynamic, and functional changes in the ELS and to assess the impact of CLN8 deficiency on axodendritic development. Results CLN8 knockdown increased the size of the Golgi apparatus, the number of mobile vesicles, and the speed of endo-lysosomes. Using the fluorescent fusion protein mApple-LAMP1-pHluorin, we detected significant lysosomal alkalization in CLN8-deficient cells. In turn, experiments in primary rat hippocampal neurons showed that CLN8 deficiency decreased the complexity and size of the somatodendritic compartment. Conclusions Our results suggest the participation of CLN8 in vesicular distribution, lysosomal pH, and normal development of the dendritic tree. We speculate that the defects triggered by CLN8 deficiency on ELS structure and dynamics underlie morphological alterations in neurons, which ultimately lead to the characteristic neurodegeneration observed in this NCL. Significance This is, to our knowledge, the first characterization of the effects of CLN8 dysfunction on the structure and dynamics of the ELS. Moreover, our findings suggest a novel role for CLN8 in somatodendritic development, which may account at least in part for the neuropathological manifestations associated with CLN8 disease. This article is protected by copyright. All rights reserved.

In Alzheimer's disease (AD), tau-protein undergoes a multi-step process involving the transition from a natively unfolded monomer to large, aggregated structures such as neurofibrillary tangles (NFTs). However, it is not yet clear which events initiate the early preclinical phase of AD tauopathy and whether they have impact on the propagation of tau pathology in later disease stages. To address this question, we analyzed the distribution of tau species phosphorylated at T231, S396/S404 and S202/T205, conformationally modified at the MC1 epitope and fibrillary tau detected by the Gallyas method (Gallyas-tau), in the brains of 15 symptomatic and 20 asymptomatic cases with AD pathology as well as of 19 nonAD cases. As initial tau lesions, we identified phosphorylated-T231-tau diffusely distributed within the somatodendritic compartment (IC-tau) and phosphorylated-S396/pS404-tau in axonal lesions of the white matter and in the neuropil (IN-tau). The subcellular localization of pT231-tau in the cell body and pS396/pS404-tau in the presynapse was confirmed in hP301L mutant Drosophila larvae. Phosphorylated-S202/T205-tau, MC1-tau and Gallyas-tau were negative for these lesions. IC- and IN-tau were observed in all analyzed regions of the human brain, including early affected regions in nonAD cases (entorhinal cortex) and late affected regions in symptomatic AD cases (cerebellum), indicating that tau pathology initiation follows similar processes when propagating into previously unaffected regions. Furthermore, a sequence of AD-related maturation of tau-aggregates was observed, initiated by the appearance of IC- and IN-tau, followed by the formation of pretangles exhibiting pT231-tau, pS396/pS404-tau and pS202/pT205-tau, then by MC1-conformational tau, and, finally, by the formation of Gallyas-positive NFTs. Since cases classified as nonAD [Braak NFT stages

During early stages of ischemic brain injury, glutamate receptor hyperactivation mediates neuronal death via osmotic cell swelling. Here we show that ischemia and excess NMDA receptor activation – conditions that trigger neuronal swelling -- cause actin filaments to undergo a rapid and extensive reorganization within the somatodendritic compartment. Normally, F-actin is concentrated within dendritic spines, with relatively little F-actin in the dendrite shaft. However, beginning in vitro, and in mouse brain after photothrombotic stroke in vivo. Following transient, sub-lethal NMDA exposure these actin changes spontaneously reverse within 1-2 hours. A combination of Na+, Cl-, water, and Ca2+ entry are all necessary, but not individually sufficient, for induction of actinification. Spine F-actin depolymerization is also required. Actinification is driven by activation of the F-actin polymerization factor inverted formin-2 (INF2). Silencing of INF2 renders neurons more vulnerable to NMDA-induced membrane leakage and cell death, and formin inhibition markedly increases ischemic infarct severity in vivo. These results show that ischemia-induced actin filament reorganization within the dendritic compartment is an intrinsic pro-survival response that protects neurons from death induced by swelling.

The molecular mechanisms underlying neuropathic pain (NP) remain poorly understood. Emerging evidence has suggested the role of microRNAs (miRNAs) in the initiation and development of NP, but the specific effects of miRNAs in NP are largely unknown. Here, we use network‐ and pathway‐based methods to investigate NP‐induced miRNA changes and their biological functions by conducting a systematic search through multiple electronic databases. Thirty‐seven articles meet the inclusion criteria. Venn analysis and target gene forecasting are performed and the results indicate that 167 overlapping target genes are co‐regulated by five down‐regulated miRNAs (rno‐miR‐183, rno‐miR‐96, rno‐miR‐30b, rno‐miR‐150 and rno‐miR‐206). Protein‐protein interaction network analysis shows that 77 genes exhibit interactions, with cyclic adenosine monophosphate (cAMP)‐dependent protein kinase catalytic subunit beta (degree = 11) and cAMP‐response element binding protein 1 (degree = 10) having the highest connectivity degree. Gene ontology analysis shows that these target genes are enriched in neuron part, neuron projection, somatodendritic compartment and nervous system development. Moreover, analysis of Kyoto Encyclopedia of Genes and Genomes reveals that three pathways, namely, axon guidance, circadian entrainment and insulin secretion, are significantly enriched. In addition, rno‐miR‐183, rno‐miR‐96, rno‐miR‐30b, rno‐miR‐150 and rno‐miR‐206 are consistently down‐regulated in the NP models, thus constituting the potential biomarkers of this disease. Characterizing these miRNAs and their target genes paves way for their future use in clinical practice.

The deposition of tau pathology in Alzheimer’s disease (AD) may occur first in axons of neurons and then progress back into the cell bodies to form neurofibrillary tangles, however, studies have not directly analyzed this relationship in relatively discrete circuits within the human hippocampus. In the early phases of tau deposition, both AT8 phosphorylation and exposure of the amino terminus of tau occurs in tauopathies, and these modifications are linked to mechanisms of synaptic and axonal dysfunction. Here, we examined the localization of these tau pathologies in well-characterized post-mortem human tissue samples from the hippocampus of 44 cases ranging between non-demented and mild cognitively impaired to capture a time at which intrahippocampal pathways show a range in the extent of tau deposition. The tissue sections were analyzed for AT8 (AT8 antibody), amino terminus exposure (TNT2 antibody), and amyloid-β (MOAB2 antibody) pathology in hippocampal strata containing the axons and neuronal cell bodies of the CA3-Schaffer collateral and dentate granule-mossy fiber pathways. We show that tau pathology first appears in the axonal compartment of affected neurons in the absence of observable tau pathology in the corresponding cell bodies in several cases. Additionally, deposition of tau in these intrahippocampal pathways was independent of the presence of Aβ plaques. We confirmed that the majority of tau pathology positive neuropil threads were axonal in origin and not dendritic using an axonal marker (i.e. SMI312 antibody) and somatodendritic marker (i.e. MAP2 antibody). Taken together, these results support the hypothesis that AT8 phosphorylation and amino terminus exposure are early pathological events and that the deposition of tau pathology, at least in the studied pathways, occurs first in the axonal compartment prior to observable pathology in the somata. These findings highlight the importance on targeting tau deposition, ideally in the initial phases of its deposition in axons. Electronic supplementary material The online version of this article (10.1186/s40478-019-0675-9) contains supplementary material, which is available to authorized users.

Microtubule-associated protein tau forms insoluble neurofibrillary tangles (NFTs), which is one of the major histopathological hallmarks of Alzheimer’s disease (AD). Many studies have demonstrated that tau causes early functional deficits prior to the formation of neurofibrillary aggregates. The redistribution of tau from axons to the somatodendritic compartment of neurons and dendritic spines causes synaptic impairment, and then leads to the loss of synaptic contacts that correlates better with cognitive deficits than amyloid-β (Aβ) aggregates do in AD patients. In this review, we discuss the underlying mechanisms by which tau is mislocalized to dendritic spines and contributes to synaptic dysfunction in AD. We also discuss the synergistic effects of tau and oligomeric forms of Aβ on promoting synaptic dysfunction in AD.

Spinocerebellar ataxia type 3 (SCA3/MJD) is caused by CAG expansion mutation resulting in a long polyQ domain in mutant ataxin-3. The mutant protein is a special type of protease, deubiquitinase, which may indicate its prominent impact on the regulation of cellular proteins levels and activity. Yet, the global model picture of SCA3 disease progression on the protein level, molecular pathways in the brain, and neurons, is largely unknown. Here, we investigated the molecular SCA3 mechanism using an interdisciplinary research paradigm combining behavioral and molecular aspects of SCA3 in the knock-in ki91 model. We used the behavior, brain magnetic resonance imaging (MRI) and brain tissue examination to correlate the disease stages with brain proteomics, precise axonal proteomics, neuronal energy recordings, and labeling of vesicles. We have demonstrated that altered metabolic and mitochondrial proteins in the brain and the lack of weight gain in Ki91 SCA3/MJD mice is reflected by the failure of energy metabolism recorded in neonatal SCA3 cerebellar neurons. We have determined that further, during disease progression, proteins responsible for metabolism, cytoskeletal architecture, vesicular, and axonal transport are disturbed, revealing axons as one of the essential cell compartments in SCA3 pathogenesis. Therefore we focus on SCA3 pathogenesis in axonal and somatodendritic compartments revealing highly increased axonal localization of protein synthesis machinery, including ribosomes, translation factors, and RNA binding proteins, while the level of proteins responsible for cellular transport and mitochondria was decreased. We demonstrate the accumulation of axonal vesicles in neonatal SCA3 cerebellar neurons and increased phosphorylation of SMI-312 positive adult cerebellar axons, which indicate axonal dysfunction in SCA3. In summary, the SCA3 disease mechanism is based on the broad influence of mutant ataxin-3 on the neuronal proteome. Processes central in our SCA3 model include disturbed localization of proteins between axonal and somatodendritic compartment, early neuronal energy deficit, altered neuronal cytoskeletal structure, an overabundance of various components of protein synthesis machinery in axons.

Cerebral deposition of abnormally aggregated α-synuclein (αSyn) is a neuropathological hallmark of Parkinson’s disease (PD). PD-associated αSyn (αSynPD) aggregates can act as proteinaceous nuclei (“seeds”) able of self-templated propagation. Since this is strikingly reminiscent to properties of proteinaceous infectious particles (prions), lessons learned from prion diseases suggest to test whether transferred αSynPD can propagate and induce neurological impairments or disease in a new host. Two studies that addressed this question provided divergent results. Intracerebral (i.c.) injection of Lewy body extracts from PD patients caused cerebral αSyn pathology, as well as nigrostriatal neurodegeneration, of wild-type mice and macaques, with the mice also showing motor impairments (Recasens et al. 2014, Ann Neurol 75:351–362). In contrast, i.c. transmission of homogenates from PD brains did not stimulate, after “> 360” days post-injection (dpi), pathological αSyn conversion or clinical symptoms in transgenic TgM83+/− mice hemizygously expressing mutated (A53T) human αSyn (Prusiner et al. 2015, PNAS 112:E5308–E5317). To advance the assessment of possible αSynPD hazards by providing further data, we examined neuropathological and clinical effects upon i.c. transmission of brain, stomach wall and muscle tissue as well as blood from PD patients in TgM83+/− mice up to 612 dpi. This revealed a subtle, yet distinctive stimulation of localized αSyn aggregation in the somatodendritic compartment and dystrophic neurites of individual or focally clustered cerebral neurons after challenge with brain and stomach wall homogenates. No such effect was observed with transmitted blood or homogenized muscle tissue. The detected stimulation of αSyn aggregation was not accompanied by apparent motor impairments or overt neurological disease in TgM83+/− mice. Our study substantiated that transmitted αSynPD seeds, including those from the stomach wall, are able to propagate in new mammalian hosts. The consequences of such propagation and potential safeguards need to be further investigated.

International audience; By limiting protein exchange between the soma and the axon, the axon initial segment (AIS) enables the segregation of specific proteins and hence the differentiation of the somatodendritic compartment and the axonal compartment. Electron microscopy and super-resolution fluorescence imaging have provided important insights in the ultrastructure of the AIS. Yet, the full extent of its filtering properties is not fully delineated. In particular, it is unclear whether and how the AIS opposes the free exchange of soluble proteins. Here we describe a robust framework to combine whole-cell photobleaching and retrospective high-resolution imaging in developing neurons. With this assay, we found that cytoplasmic soluble proteins that are not excluded from the axon upon expression over tens of hours exhibit a strong mobility reduction at the AIS-i.e., are indeed compartmentalized-when monitored over tens of minutes. This form of compartmentalization is developmentally regulated, requires intact F-actin and may be correlated with the composition and ultrastructure of the submembranous spectrin cytoskeleton. Using computational modeling, we provide evidence that both neuronal morphology and the AIS regulate this compartmentalization but act on distinct time scales, with the AIS having a more pronounced effect on fast exchanges. Our results thus suggest that the rate of protein accumulation in the soma may impact to what extent and over which timescales freely moving molecules can be segregated from the axon. This in turn has important implications for our understanding of compartment-specific signaling in neurons.

Increasingly, research suggests that neurodegenerative diseases and dementias are caused not by unique, solitary cellular mechanisms, but by multiple contributory mechanisms manifesting as heterogeneous clinical presentations. However, diverse neurodegenerative diseases also share common pathological hallmarks and cellular mechanisms. One such mechanism involves the redistribution of the microtubule associated protein tau from the axon into the somatodendritic compartment of neurons, followed by the mislocalization of tau into dendritic spines, resulting in postsynaptic functional deficits. Here we review various signaling pathways that trigger the redistribution of tau to the cell body and dendritic tree, and its mislocalization to dendritic spines. The convergence of multiple pathways in different disease models onto this final common pathway suggests that it may be an attractive pathway to target for developing new treatments for neurodegenerative diseases.

Abtract Tau is a microtubule‐associated protein (MAP) that is localized to the axon. In Alzheimer's disease (AD), the distribution of tau undergoes a remarkable alteration, leading to the formation of tau inclusions in the somatodendritic compartment. While the abnormal aggregated tau has been extensively studied in human patient tissues and animal models of AD, how normal tau localizes to the axon, which would be the foundation to understand how the mis‐localization occurs, has not been well studied due to the poor detectability of normal unaggregated tau in vivo. Therefore, we developed immunohistochemical techniques that can detect normal mouse and human tau in brain tissues with high sensitivity. Using these techniques, we demonstrate the global distribution of tau in the mouse brain and confirmed that normal tau is exclusively localized to the axonal compartment in vivo. Interestingly, tau antibodies strongly labeled nonmyelinated axons such as hippocampal mossy fibers, while white matters generally exhibited low levels of immunoreactivity. Furthermore, mouse tau is highly expressed not only in neurons but also in oligodendrocytes. With super resolution imaging using the stimulated‐depletion microscopy, axonal tau appeared punctate rather than fibrous, indicating that tau decorates microtubules sparsely. Co‐labeling with presynaptic and postsynaptic markers revealed that normal tau is not localized to synapses but sparsely distributes in the axon. Taken together, this study reports novel antibodies to investigate the localization and mis‐localization of tau in vivo and novel findings of normal tau localization in the mouse brain.

Key points GABAA receptors have been described in the axonal compartment of neurons; contrary to dendritic GABAA receptors, axonal GABAA receptors usually induce depolarizing responses. In this study we describe the presence of functional axonal GABAA receptors in cerebellar Purkinje cells by using a combination of direct patch-clamp recordings from the axon terminals and laser GABA photolysis. In Purkinje cells, axonal GABAA receptors are depolarizing and induce an increase in neurotransmitter release that results in a change of short-term synaptic plasticity. These results contribute to our understanding of the cellular mechanisms of action of axonal GABAA receptors and highlight the importance of the presynaptic compartment in neuronal computation. Abstract In neurons of the adult brain, somatodendritic GABAA receptors (GABAA Rs) mediate fast synaptic inhibition and play a crucial role in synaptic integration. GABAA Rs are not only present in the somatodendritic compartment, but also in the axonal compartment where they modulate action potential (AP) propagation and transmitter release. Although presynaptic GABAA Rs have been reported in various brain regions, their mechanisms of action and physiological roles remain obscure, particularly at GABAergic boutons. Here, using a combination of direct whole-bouton or perforated patch-clamp recordings and local GABA photolysis in single axonal varicosities of cerebellar Purkinje cells, we investigate the subcellular localization and functional role of axonal GABAA Rs both in primary cultures and acute slices. Our results indicate that presynaptic terminals of PCs carry GABAA Rs that behave as auto-receptors; their activation leads to a depolarization of the terminal membrane after an AP due to the relatively high cytoplasmic Cl- concentration in the axon, but they do not modulate the AP itself. Paired recordings from different terminals of the same axon show that the GABAA R-mediated local depolarizations propagate substantially to neighbouring varicosities. Finally, the depolarization mediated by presynaptic GABAA R activation augmented Ca2+ influx and transmitter release, resulting in a marked effect on short-term plasticity. Altogether, our results reveal a mechanism by which presynaptic GABAA Rs influence neuronal computation.

Dendritic spines are postsynaptic structures in neurons often having a mushroom-like shape. Physiological significance and cytoskeletal mechanisms that maintain this shape are poorly understood. The spectrin-based membrane skeleton maintains the biconcave shape of erythrocytes, but whether spectrins also determine the shape of nonerythroid cells is less clear. We show that βIII spectrin in hippocampal and cortical neurons from rodent embryos of both sexes is distributed throughout the somatodendritic compartment but is particularly enriched in the neck and base of dendritic spines and largely absent from spine heads. Electron microscopy revealed that βIII spectrin forms a detergent-resistant cytoskeletal network at these sites. Knockdown of βIII spectrin results in a significant decrease in the density of dendritic spines. Surprisingly, the density of presynaptic terminals is not affected by βIII spectrin knockdown. However, instead of making normal spiny synapses, the presynaptic structures in βIII spectrin-depleted neurons make shaft synapses that exhibit increased amplitudes of miniature EPSCs indicative of excessive postsynaptic excitation. Thus, βIII spectrin is necessary for formation of the constricted shape of the spine neck, which in turn controls communication between the synapse and the parent dendrite to prevent excessive excitation. Notably, mutations of SPTNB2 encoding βIII spectrin are associated with neurodegenerative syndromes, spinocerebellar ataxia Type 5, and spectrin-associated autosomal recessive cerebellar ataxia Type 1, but molecular mechanisms linking βIII spectrin functions to neuronal pathologies remain unresolved. Our data suggest that spinocerebellar ataxia Type 5 and spectrin-associated autosomal recessive cerebellar ataxia Type 1 pathology likely arises from poorly controlled synaptic activity that leads to excitotoxicity and neurodegeneration.SIGNIFICANCE STATEMENT Dendritic spines are small protrusions from neuronal dendrites that make synapses with axons of other neurons in the brain. Dendritic spines usually have a mushroom-like shape, which is essential for brain functions, because aberrant spine morphology is associated with many neuropsychiatric disorders. The bulbous head of a mushroom-shaped spine makes the synapse, whereas the narrow neck transmits the incoming signals to the dendrite and supposedly controls the signal propagation. We show that a cytoskeletal protein βIII spectrin plays a key role for the formation of narrow spine necks. In the absence of βIII spectrin, dendritic spines collapse onto dendrites. As a result, synaptic strength exceeds acceptable levels and damages neurons, explaining pathology of human syndromes caused by βIII spectrin mutations.

Tau is a microtubule-associated protein, localizing mainly in the axon of mature neurons. Phenotypic analysis of Tau knockout mice has revealed an impairment of synaptic plasticity but without gross changes in brain morphology. Since we previously described the presence of tau mRNA in the somatodendritic compartment, including the postsynapse, and demonstrated that it could be locally translated in response to glutamate, it appears that the regulated translation of synaptic tau can have a direct impact on synaptic function. Using SH-SY5Y cells, we herein confirm that glutamate dose-dependently regulates the translation of tau protein without altering tau mRNA levels. This is supported by the finding that cycloheximide blocks glutamate-stimulated increases in tau protein levels. Our observation that neural excitation can directly upregulate tau mRNA translation helps explain the pathological accumulation of tau in the somatodendrite.

Tau is a microtubule (MT)-associated protein that is localized to the axon. In Alzheimer's disease, the distribution of tau undergoes a remarkable alteration, leading to the formation of tau inclusions in the somatodendritic compartment. To investigate how this mislocalization occurs, we recently developed immunohistochemical tools that can separately detect endogenous mouse and exogenous human tau with high sensitivity, which allows us to visualize not only the pathological but also the pre-aggregated tau in mouse brain tissues of both sexes. Using these antibodies, we found that in tau-transgenic mouse brains, exogenous human tau was abundant in dendrites and somata even in the presymptomatic period, whereas the axonal localization of endogenous mouse tau was unaffected. In stark contrast, exogenous tau was properly localized to the axon in human tau knock-in mice. We tracked this difference to the temporal expression patterns of tau. Endogenous mouse tau and exogenous human tau in human tau knock-in mice exhibited high expression levels during the neonatal period and strong suppression into the adulthood. However, human tau in transgenic mice was expressed continuously and at high levels in adult animals. These results indicated the uncontrolled expression of exogenous tau beyond the developmental period as a cause of mislocalization in the transgenic mice. Superresolution microscopic and biochemical analyses also indicated that the interaction between MTs and exogenous tau was impaired only in the tau-transgenic mice, but not in knock-in mice. Thus, the ectopic expression of tau may be critical for its somatodendritic mislocalization, a key step of the tauopathy. SIGNIFICANCE STATEMENT Somatodendritic localization of tau may be an early step leading to the neuronal degeneration in tauopathies. However, the mechanisms of the normal axonal distribution of tau and the mislocalization of pathological tau remain obscure. Our immunohistochemical and biochemical analyses demonstrated that the endogenous mouse tau is transiently expressed in neonatal brains, that exogenous human tau expressed corresponding to such tau expression profile can distribute into the axon, and that the constitutive expression of tau into adulthood (e.g., human tau in transgenic mice) results in abnormal somatodendritic localization. Thus, the expression profile of tau is tightly associated with the localization of tau, and the ectopic expression of tau in matured neurons may be involved in the pathogenesis of tauopathy.

In many neuronal types, axon initial segment (AIS) geometry critically influences neuronal excitability. Interestingly, the axon of rat SNc dopaminergic (DA) neurons displays a highly variable location and most often arises from an axon-bearing dendrite (ABD). We combined current-clamp somatic and dendritic recordings, outside-out recordings of dendritic sodium and potassium currents, morphological reconstructions and multicompartment modeling on male and female rat SNc DA neurons to determine cell-to-cell variations in AIS and ABD geometry, and their influence on neuronal output (spontaneous pacemaking frequency, action potential [AP] shape). Both AIS and ABD geometries were found to be highly variable from neuron to neuron. Surprisingly, we found that AP shape and pacemaking frequency were independent of AIS geometry. Modeling realistic morphological and biophysical variations helped us clarify this result: in SNc DA neurons, the complexity of the ABD combined with its excitability predominantly define pacemaking frequency and AP shape, such that large variations in AIS geometry negligibly affect neuronal output and are tolerated. SIGNIFICANCE STATEMENT In many neuronal types, axon initial segment (AIS) geometry critically influences neuronal excitability. In the current study, we describe large cell-to-cell variations in AIS length or distance from the soma in rat substantia nigra pars compacta dopaminergic neurons. Using neuronal reconstruction and electrophysiological recordings, we show that this morphological variability does not seem to affect their electrophysiological output, as neither action potential properties nor pacemaking frequency is correlated with AIS morphology. Realistic multicompartment modeling suggests that this robustness to AIS variation is mainly explained by the complexity and excitability of the somatodendritic compartment.

Selective cargo transport into axons and dendrites over the microtubule network is essential for neuron polarization. The axon initial segment (AIS) separates the axon from the somatodendritic compartment and controls the microtubule-dependent transport into the axon. Interestingly, the AIS has a characteristic microtubule organization; it contains bundles of closely spaced microtubules with electron dense cross-bridges, referred to as microtubule fascicles. The microtubule binding protein TRIM46 localizes to the AIS and when overexpressed in non-neuronal cells forms microtubule arrays that closely resemble AIS fascicles in neurons. However, the precise role of TRIM46 in microtubule fasciculation in neurons has not been studied. Here we developed a novel correlative light and electron microscopy approach to study AIS microtubule organization. We show that in cultured rat hippocampal neurons of both sexes, TRIM46 levels steadily increase at the AIS during early neuronal differentiation and at the same time closely spaced microtubules form, whereas the fasciculated microtubules appear at later developmental stages. Moreover, we localized TRIM46 to the electron dense cross-bridges and show that depletion of TRIM46 causes loss of cross-bridges and increased microtubule spacing. These data indicate that TRIM46 has an essential role in organizing microtubule fascicles in the AIS. SIGNIFICANCE STATEMENT The axon initial segment (AIS) is a specialized region at the proximal axon where the action potential is initiated. In addition the AIS separates the axon from the somatodendritic compartment, where it controls protein transport to establish and maintain neuron polarity. Cargo vesicles destined for the axon recognize specialized microtubule tracks that enter the AIS. Interestingly the microtubules entering the AIS form crosslinked bundles, called microtubule fascicules. Recently we found that the microtubule-binding protein TRIM46 localizes to the AIS, where it may organize the AIS microtubules. In the present study we developed a novel correlative light and electron microscopy approach to study the AIS microtubules during neuron development and identified an essential role for TRIM46 in microtubule fasciculation.

The microtubule associated protein tau is mainly found in the cell’s cytosol but recently it was also shown in the extracellular space. In neurodegenerative diseases, like Alzheimer’s disease (AD), pathological tau spreads from neuron to neuron enhancing neurodegeneration. Here, we show that HEK293 cells and neurons in culture uptake extracellular normal and pathological Tau. Muscarinic receptor antagonists atropine and pirenzepine block 80% this uptake. CHO cells do not express these receptors therefore cannot uptake tau, unless transfected with M1 and/or M3 receptor. These results strongly suggest that muscarinic receptors mediate this process. Uptake of normal tau in neurons enhances neuronal process formation but a pseudophosphorylated form of tau (pathological human tau, PH-Tau) disrupts them and accumulates in the somatodendritic compartment. AD hyperphosphorylated tau (AD P-Tau) has similar effects as PH-Tau on cultured neurons. Addition of either PH-Tau or AD P-tau to neuronal cultures induced microglial activation. In conclusion, uptake of extracellular tau is mediated by muscarinic receptors with opposite effects: normal tau stabilizes neurites; whereas pathological tau disrupts this process leading to neurodegeneration.

Sortilin is a member of the vacuolar protein sorting 10 protein (VPS10P) domain receptor family, which carries out signal transduction and protein transport in cells. Sortilin serves as the third, G-protein uncoupled, receptor of neurotensin that can modulate various brain functions. More recent data indicate an involvement of sortilin in mood disorders, dementia and Alzheimer-type neuropathology. However, data regarding the normal pattern of regional and cellular expression of sortilin in the human brain are not available to date. Using postmortem adult human brains free of neuropathology, the current study determined sortilin immunoreactivity (IR) across the entire brain. Sortilin IR was broadly present in the cerebrum and subcortical structures, localizing to neurons in the somatodendritic compartment, but not to glial cells. In the cerebrum, sortilin IR exhibited differential regional and laminar patterns, with pyramidal, multipolar and polymorphic neurons in cortical layers II–VI, hippocampal formation and amygdaloid complex more distinctly labeled relative to GABAergic interneurons. In the striatum and thalamus, numerous small-to-medium sized neurons showed light IR, with a small group of large sized neurons heavily labeled. In the midbrain and brainstem, sortilin IR was distinct in neurons at the relay centers of descending and ascending neuroanatomical pathways. Dopaminergic neurons in the substantia nigra, cholinergic neurons in the basal nuclei of Meynert and noradrenergic neurons in the locus coeruleus co-expressed strong sortilin IR in double immunofluorescence. In comparison, sortilin IR was weak in the olfactory bulb and cerebellar cortex, with the mitral and Purkinje cells barely visualized. A quantitative analysis was carried out in the lateral, basolateral, and basomedial nuclei of the amygdaloid complex, as well as cortical layers II–VI, which established a positive correlation between the somal size and the intensity of sortilin IR among labeled neurons. Together, the present study demonstrates a predominantly neuronal expression of sortilin in the human brain with substantial regional and cell-type variability. The enriched expression of sortilin in pyramidal, dopaminergic, noradrenergic and cholinergic neurons suggests that this protein may be particularly required for signal transduction, protein trafficking and metabolic homeostasis in populations of relatively large-sized projective neurons.

Sensory neurons perceive environmental cues and are important of organismal survival. Peripheral sensory neurons interact intimately with glial cells. While the function of axonal ensheathment by glia is well studied, less is known about the functional significance of glial interaction with the somatodendritic compartment of neurons. Herein, we show that three distinct glia cell types differentially wrap around the axonal and somatodendritic surface of the polymodal dendritic arborization (da) neuron of the Drosophila peripheral nervous system for detection of thermal, mechanical, and light stimuli. We find that glial cell-specific loss of the chromatin modifier gene dATRX in the subperineurial glial layer leads to selective elimination of somatodendritic glial ensheathment, thus allowing us to investigate the function of such ensheathment. We find that somatodendritic glial ensheathment regulates the morphology of the dendritic arbor, as well as the activity of the sensory neuron, in response to sensory stimuli. Additionally, glial ensheathment of the neuronal soma influences dendritic regeneration after injury.

The expression of the endocannabinoid (eCB) system, including cannabinoid receptor type 1 (CB1R) and the cannabinoid synthesizing (NAPE-PLD) and degrading (FAAH) enzymes, has been well-characterized in the retina of rodents and monkeys. More recently, the presence of CB1R was localized throughout the dorsal lateral geniculate nucleus of the thalamus of vervet monkeys. Given that the retina projects also to the pulvinar either via a direct projection or via the superior colliculus, it was reasonable to assume that this system would be present therein. The visual pulvinar, namely the inferior pulvinar (PI) region, was delineated with calbindin immunohistochemical staining. Using Western blots and immunofluorescence, we demonstrated that CB1R, NAPE-PLD and FAAH are expressed in the PI of the vervet monkey. Throughout the PI, CB1R was mainly colocalized with VGLUT2-positive axon terminals in the vicinity of calbindin and parvalbumin-positive neurons. NAPE-PLD and FAAH rather colocalized with calbindin over the somatodendritic compartment of PI neurons. Our results suggest that visual information coming from the retina and entering the PI is modulated by the eCB system on its way to the dorsal visual stream. These results provide insights for understanding the role of eCBs in the modulation of visual thalamic inputs and, hence, visual perception.

In mammalian neurons, the axon initial segment (AIS) electrically connects the somatodendritic compartment with the axon and converts the incoming synaptic voltage changes into a temporally precise action potential (AP) output code. Although axons often emanate directly from the soma, they may also originate more distally from a dendrite, the implications of which are not well-understood. Here, we show that one-third of the thick-tufted layer 5 pyramidal neurons have an axon originating from a dendrite and are characterized by a reduced dendritic complexity and thinner main apical dendrite. Unexpectedly, the rising phase of somatic APs is electrically indistinguishable between neurons with a somatic or a dendritic axon origin. Cable analysis of the neurons indicated that the axonal axial current is inversely proportional to the AIS distance, denoting the path length between the soma and the start of the AIS, and to produce invariant somatic APs, it must scale with the local somatodendritic capacitance. In agreement, AIS distance inversely correlates with the apical dendrite diameter, and model simulations confirmed that the covariation suffices to normalize the somatic AP waveform. Therefore, in pyramidal neurons, the AIS location is finely tuned with the somatodendritic capacitive load, serving as a homeostatic regulation of the somatic AP in the face of diverse neuronal morphologies.

Neurons represent the cellular substrate for information processing in the nervous system. Already around 1900 the Spanish neuroanatomist Ramon y Cajal proposed that neurons possess two discrete functional domains, the axonal and the somatodendritic compartment. Cajal established the foundation for the neuron doctrine by suggesting dendrites to be the synaptic input regions of neurons, and that information processing travels from dendritic regions towards axon terminals and output synapses (“the theory of dynamic polarization”, Shepherd, 1991). Despite a number of exceptions, for most neurons this rule prevails to the present. Therefore, dendritic architecture has two fundamental functions in the nervous system. First dendrites expand the receptive surface of neurons, and their shape dictates how many and which presynaptic neurons can contact a postsynaptic dendritic arbor. Thus, dendritic structure influences the number of synapses as well as the wiring logic within neuronal networks. Second dendritic structure impacts the temporal and spatial integration of postsynaptic potentials. Accordingly, in different types of neurons with different functions dendritic gestalt differs significantly, and dendritic architecture often serves to classify neuron types. In most cases, however, the specific function of dendritic architecture remains largely elusive. Dendritic structure analysis is further bedeviled by dendrites exhibiting voltage-gated ion channels which themselves vastly modify function and computing power. Although a multitude of neurodevelopmental and neurodegenerative disorders coincides with dendritic defects, it often remains unclear whether these structural defects are the cause or a consequence of the dysfunction. Therefore, on the one hand it is important to determine the contribution of dendritic structure to the function of different types of healthy neurons. On the other hand the question arises whether dendritic defects impact neuronal function qualitatively and to what degree of dendritic defect neuronal function can be maintained. This article will first summarize basic functions of passive dendritic architecture which applies for most neurons but confers variable characteristics to different types of neurons. It will be discussed how the location of input synapses in a passive electrical structure affects the integration of postsynaptic potentials. Then principles will be introduced how this localization-dependence of synaptic inputs into dendrites can be compensated for. And finally, an identified Drosophila motoneuron will serve as an example that at least in specific types of neurons basic function can be maintained with a minimum number of dendrites and input synapses. By contrast, in this example dendritic structure is imperative for fine tuning of adaptive behavioral functions which are essential for survival and reproduction. These findings will then be discussed in the context of other neuronal functions.

Dopamine (DA) transmission is governed by processes that regulate release from axonal boutons in the forebrain and the somatodendritic compartment inmidbrain, and by clearance by the DA transporter, diffusion, and extracellular metabolism. Wereview how axonal DA release is regulated by neuronal activity and by autoreceptors and heteroreceptors, and address how quantal release events are regulated in size and frequency. In brain regions densely innervated by DA axons, DA clearance is due predominantly to uptake by the DA transporter, whereas in cortex, midbrain, and other regions with relatively sparse DA inputs, the norepinephrine transporter and diffusion are involved. We discuss the role of DA uptake in restricting the sphere of influence of DA and in temporal accumulation of extracellular DA levels upon successive action potentials. The tonic discharge activity of DA neurons may be translated into a tonicextracellular DA level, whereas their bursting activity can generate discrete extracellular DA transients.

Autophagy is an essential degradative pathway that maintains neuronal homeostasis and prevents axon degeneration. Initial observations suggest that autophagy is spatially regulated in neurons, but how autophagy is regulated in distinct neuronal compartments is unclear. Using live-cell imaging in mouse hippocampal neurons, we establish the compartment-specific mechanisms of constitutive autophagy under basal conditions, as well as in response to stress induced by nutrient deprivation. We find that at steady state, the cell soma contains populations of autophagosomes derived from distinct neuronal compartments and defined by differences in maturation state and dynamics. Axonal autophagosomes enter the soma and remain confined within the somatodendritic domain. This compartmentalization likely facilitates cargo degradation by enabling fusion with proteolytically active lysosomes enriched in the soma. In contrast, autophagosomes generated within the soma are less mobile and tend to cluster. Surprisingly, starvation did not induce autophagy in either the axonal or somatodendritic compartment. While starvation robustly decreased mTORC1 signaling in neurons, this decrease was not sufficient to activate autophagy. Furthermore, pharmacological inhibition of mammalian target of rapamycin with Torin1 also was not sufficient to markedly upregulate neuronal autophagy. These observations suggest that the primary physiological function of autophagy in neurons may not be to mobilize amino acids and other biosynthetic building blocks in response to starvation, in contrast to findings in other cell types. Rather, constitutive autophagy in neurons may function to maintain cellular homeostasis by balancing synthesis and degradation, especially within distal axonal processes far removed from the soma.SIGNIFICANCE STATEMENTAutophagy is an essential homeostatic process in neurons, but neuron-specific mechanisms are poorly understood. Here, we compare autophagosome dynamics within neuronal compartments. Axonal autophagy is a vectorial process that delivers cargo from the distal axon to the soma. The soma, however, contains autophagosomes at different maturation states, including input received from the axon combined with locally generated autophagosomes. Once in the soma, autophagosomes are confined to the somatodendritic domain, facilitating cargo degradation and recycling of biosynthetic building blocks to primary sites of protein synthesis. Neuronal autophagy is not robustly upregulated in response to starvation or mammalian target of rapamycin inhibition, suggesting that constitutive autophagy in neurons maintains homeostasis by playing an integral role in regulating the quality of the neuronal proteome.

Neurons are highly polarized cells that contain specialized subcellular domains involved in information transmission in the nervous system. Specifically, the somatodendritic compartment receives neuronal inputs while the axons convey information through the synapse. The establishment of asymmetric domains requires a specific delivery of components, including organelles, proteins, and membrane. The Rab family of small GTPases plays an essential role in membrane trafficking. Signaling cascades triggered by extrinsic and intrinsic factors tightly regulate Rab functions in cells, with Rab protein activation depending on GDP/GTP binding to establish a binary mode of action. This review summarizes the contributions of several Rab family members involved in trans-Golgi, early/late endosomes, and recycling endosomes during neurite development and axonal outgrowth. The regulation of some Rabs by guanine exchanging factors and GTPase activating proteins will also be addressed. Finally, discussion will be provided on how specific effector-mediated Rab activation modifies several molecules essential to neuronal differentiation. © 2016 Wiley Periodicals, Inc.

Neuroscience Inositol-triphosphate (IP3) receptors regulate the intracellular calcium concentration in the somatodendritic compartment of central neurons. Whether axons also possess functional IP3 receptors and what impact their activation might have are not known. Cerebellar Purkinje cells offer an ideal model because they contain a high level of IP3 receptors. Using chromophore tags that release IP3 when irradiated, Gomez et al. found that functional IP3 receptors are present in the entire axon. Different axon regions displayed different IP3-producing pathways, and IP3 receptor activation had different consequences depending on receptor localization. For instance, IP3 receptor activation in synaptic terminals caused neurotransmitter release, and receptor activation in the axon initial segment blocked action potential firing. IP3 receptor–linked signaling pathways may therefore be important in controlling axon functions. Proc. Natl. Acad. Sci. U.S.A. 117 , 11097 (2020).

The serotonin transporter (SERT) regulates serotonergic neurotransmission by retrieving released serotonin and replenishing vesicular stores. SERT is not only delivered to axons but it is also present on the neuronal soma and on dendrites. It has not been possible to restrict the distribution of SERT without affecting transporter function. Hence, the physiological role of somatodendritic SERT remains enigmatic. The SERT C-terminus harbors a conserved RI-motif, which recruits SEC24C, a cargo receptor in the coatomer protein-II coat. Failure to engage SEC24C precludes axonal enrichment of SERT. Here we introduced a point mutation into the RI-motif of human SERT causing confinement of the resulting - otherwise fully functional - hSERT-R607A on the somatodendritic membrane of primary rat dorsal raphe neurons. Transgenic expression of the corresponding Drosophila mutant dSERT-R599A led to its enrichment in the somatodendritic compartment of serotonergic neurons in the fly brain. We explored the possible physiological role of somatodendritic SERT by comparing flies harboring wild type SERT and dSERT-R599A in a behavioral paradigm for serotonin-modulated odor perception. When globally re-expressed in serotonergic neurons, wild type SERT but not dSERT-R599A restored ethanol preference. In contrast, dSERT-R599A restored ethanol preference after targeted expression in contralaterally projecting, serotonin-immunoreactive deuterocerebral (CSD) interneurons, while expression of wild type SERT caused ethanol aversion. We conclude that, in CSD neurons, (i) somatodendritic SERT supports ethanol attraction, (ii) axonal SERT specifies ethanol aversion, (iii) the effect of axonal SERT can override that of somatodendritic SERT. These observations demonstrate a distinct biological role of somatodendritic and axonal serotonin transport. This article is part of the issue entitled ‘Special Issue on Neurotransmitter Transporters’.

Tau is a microtubule-associated protein that is localized to the axon. In Alzheimer’s disease, the distribution of tau undergoes a remarkable alteration, leading to the formation of tau inclusions in the somatodendritic compartment. To investigate how this mis-localization occurs, we recently developed immunohistochemical techniques that can separately detect endogenous mouse and exogenous human tau with high sensitivity, which allows us to visualize not only the pathological but pre-aggregated tau in mouse brain tissues of both sex. In tau transgenic mouse brains, exogenous human tau was abundant in dendrites and somata even in the presymptomatic period, whereas the axonal localization of endogenous mouse tau was unaffected. In stark contrast, exogenous tau was properly localized to the axon in human tau knock-in mice. We tracked this difference to the temporal expression patterns of tau. Tau mRNA was continuously expressed in the transgenic mice, whereas endogenous tau and exogenous tau in the knock-in mice exhibited high expression levels during the neonatal period and strong suppression into the adulthood. These results indicated the uncontrolled expression of exogenous tau beyond the developmental period as a cause of mis-localization in the transgenic mice. Super-resolution microscopic and biochemical analyses also indicated that the interaction between microtubules and exogenous tau was indeed impaired in the tau transgenic mice. Thus, the ectopic expression of tau may be critical for its somatodendritic mis-localization, a key step of the tauopathy.Significance StatementSomatodendritic localization of tau may be an early step leading to the neuronal degeneration in tauopathies. However, the mechanisms of the normal axonal distribution of tau and the mis-localization of pathological tau remain obscure. Our immunohistochemical and biochemical analyses demonstrated that the endogenous mouse tau is transiently expressed in neonatal brains, that exogenous human tau expressed corresponding to such tau expression profile can distribute into the axon, and that the constitutive expression of tau into adulthood (ex. human tau in Tg mice) results in abnormal somatodendritic localization. Thus, the expression profile of tau is tightly associated with the localization of tau, and the ectopic expression of tau in matured neurons may be involved in the pathogenesis of tauopathy.

Neurons lose intrinsic axon regenerative ability with maturation, but the mechanism remains unclear. Using an in-vitro laser axotomy model, we show a progressive decline in the ability of cut CNS axons to form a new growth cone and then elongate. Failure of regeneration was associated with increased retraction after axotomy. Transportation into axons becomes selective with maturation; we hypothesized that selective exclusion of molecules needed for growth may contribute to regeneration decline. With neuronal maturity rab11 vesicles (which carry many molecules involved in axon growth) became selectively targeted to the somatodendritic compartment and excluded from axons by predominant retrograde transport However, on overexpression rab11 was mistrafficked into proximal axons, and these axons showed less retraction and enhanced regeneration after axotomy. These results suggest that the decline of intrinsic axon regenerative ability is associated with selective exclusion of key molecules, and that manipulation of transport can enhance regeneration., eLife digest The nerves in the brain and spinal cord can be damaged by trauma, stroke and other conditions. Damage to these nerve fibres can destroy the connections they form with each other, which may lead to paralysis, loss of sensation and loss of body control. If we could stimulate the regeneration and reconnection of the damaged nerve fibres then neurological function could be restored. However, although embryonic nerve fibres can regenerate when they are transplanted into the adult central nervous system, this regenerative ability appears to be lost as the nerve fibres mature. To investigate when and why nerve fibres lose the ability to regenerate, Koseki et al. first developed a tissue culture assay in which individual nerve fibres were cut with a laser and imaged for several hours to track their regeneration (or failure to regenerate). The results demonstrate that nerve fibres from the central nervous system progressively lose the ability to grow and regenerate as they mature. To investigate why mature nerve fibres cannot regenerate, Koseki et al. measured whether nerve fibres can transport some of the molecules needed for growth and regeneration to sites of damage. This showed that the compartments in which some key growth molecules are transported become excluded from mature nerve fibres. These compartments are marked by a protein called rab11, and Koseki et al. found that forcing rab11 back into mature nerve fibres restored their ability to regenerate. There is still a lot of work needed before these findings can lead to a new regeneration treatment for patients, but it is a crucial step forwards. Furthermore, the assay developed by Koseki et al. could be used to develop and test such treatments.

The Na+/K+/Cl- cotransporter-1 (NKCC1) and the K+/Cl- cotransporter-2 (KCC2) set the transmembrane Cl- gradient in the brain, and are implicated in epileptogenesis. We studied the postnatal distribution of NKCC1 and KCC2 in wild-type (WT) mice, and in a mouse model of sleep-related epilepsy, carrying the mutant β2-V287L subunit of the nicotinic acetylcholine receptor (nAChR). In WT neocortex, immunohistochemistry showed a wide distribution of NKCC1 in neurons and astrocytes. At birth, KCC2 was localized in neuronal somata, whereas at subsequent stages it was mainly found in the somatodendritic compartment. The cotransporters' expression was quantified by densitometry in the transgenic strain. KCC2 expression increased during the first postnatal weeks, while the NKCC1 amount remained stable, after birth. In mice expressing β2-V287L, the KCC2 amount in layer V of prefrontal cortex (PFC) was lower than in the control littermates at postnatal day 8 (P8), with no concomitant change in NKCC1. Consistently, the GABAergic excitatory to inhibitory switch was delayed in PFC layer V of mice carrying β2-V287L. At P60, the amount of KCC2 was instead higher in mice bearing the transgene. Irrespective of genotype, NKCC1 and KCC2 were abundantly expressed in the neuropil of most thalamic nuclei since birth. However, KCC2 expression decreased by P60 in the reticular nucleus, and more so in mice expressing β2-V287L. Therefore, a complex regulatory interplay occurs between heteromeric nAChRs and KCC2 in postnatal forebrain. The pathogenetic effect of β2-V287L may depend on altered KCC2 amounts in PFC during synaptogenesis, as well as in mature thalamocortical circuits.

During neuronal development, the microtubule-associated protein tau becomes enriched in the axon, where it remains concentrated in the healthy brain. In tauopathies such as Alzheimer's disease, tau redistributes from the axon to the somatodendritic compartment. However, the cellular mechanism that regulates tau's localization remains unclear. We report here that tau interacts with the Ca(2+)-regulated plasma membrane–binding protein annexin A2 (AnxA2) via tau's extreme N terminus encoded by the first exon (E1). Bioinformatics analysis identified two conserved eight-amino-acids-long motifs within E1 in mammals. Using a heterologous yeast system, we found that disease-related mutations and pseudophosphorylation of Tyr-18, located within E1 but outside of the two conserved regions, do not influence tau's interaction with AnxA2. We further observed that tau interacts with the core domain of AnxA2 in a Ca(2+)-induced open conformation and interacts also with AnxA6. Moreover, lack of E1 moderately increased tau's association rate to microtubules, consistent with the supposition that the presence of the tau–annexin interaction reduces the availability of tau to interact with microtubules. Of note, intracellular competition through overexpression of E1-containing constructs reduced tau's axonal enrichment in primary neurons. Our results suggest that the E1-mediated tau–annexin interaction contributes to the enrichment of tau in the axon and is involved in its redistribution in pathological conditions.

Neurons have developed elaborate mechanisms for sorting of proteins to their destination in dendrites and axons as well as dynamic local trafficking. Recent evidence suggests that polarized axonal sorting of β-site converting enzyme 1 (BACE1), a type I transmembrane aspartyl protease involved in Alzheimer's disease (AD) pathogenesis, entails an unusual journey. In hippocampal neurons, BACE1 internalized from dendrites is conveyed in recycling endosomes via unidirectional retrograde transport towards the soma and sorted to axons where BACE1 becomes enriched. In comparison to other transmembrane proteins that undergo transcytosis or elimination in somatodendritic compartment, vectorial transport of internalized BACE1 in dendrites is unique and intriguing. Dysfunction of protein transport contributes to pathogenesis of AD and other neurodegenerative diseases. Therefore, characterization of BACE1 transcytosis is an important addition to the multiple lines of evidence that highlight the crucial role played by endosomal trafficking pathway as well as axonal sorting mechanisms in AD pathogenesis.

Dopaminergic signaling in the reward pathway in the brain has been shown to play an important role in food intake and the development of obesity. Obese rats release less dopamine (DA) in the nucleus accumbens (NAc) after food intake, and amphetamine stimulated striatal DA release is reduced in vivo in obese subjects. These studies suggest that DA hypofunction associated with hedonic dysregulation is involved in the pathophysiology of obesity. To identify brain changes in obesity, quantitative measures of DA synaptic markers were compared in postmortem brain tissues of normal weight and obese subjects over a range of increasing body mass indices (BMI). DA transporter (DAT) numbers in the striatum were compared to the relative expression of DAT, tyrosine hydroxylase (TH) and D2 dopamine receptors (DRD2) in midbrain DA neurons. Radioligand binding assays of [3H]WIN35,428 demonstrated that the number of striatal DAT binding sites was inversely correlated with increasing BMI (r = −0.47; p < 0.01). DAT and TH gene expression were significantly decreased in the somatodendritic compartment of obese subjects (p < 0.001), with no significant change in DRD2 compared to normal weight subjects. The reduced density of striatal DAT with corresponding reductions in DAT and TH gene expression in substantia nigra (SN) suggests, that obesity is associated with hypodopaminergic function. A DA reward deficiency syndrome has been suggested to underlie abnormal eating behavior that leads to obesity. Neurobiological changes in presynaptic DA markers demonstrated postmortem in human brain support a link between hedonic DA dysregulation and obesity.

Actin, spectrin, and associated molecules form a membrane-associated periodic skeleton (MPS) in neurons. In the MPS, short actin filaments, capped by actin-capping proteins, form ring-like structures that wrap around the circumference of neurites, and these rings are periodically spaced along the neurite by spectrin tetramers, forming a quasi-1D lattice structure. This 1D MPS structure was initially observed in axons and exists extensively in axons, spanning nearly the entire axonal shaft of mature neurons. Such 1D MPS was also observed in dendrites, but the extent to which it exists and how it develops in dendrites remain unclear. It is also unclear whether other structural forms of the membrane skeleton are present in neurons. Here, we investigated the spatial organizations of spectrin, actin, and adducin, an actin-capping protein, in the dendrites and soma of cultured hippocampal neurons at different developmental stages, and compared results with those obtained in axons, using superresolution imaging. We observed that the 1D MPS exists in a substantial fraction of dendritic regions in relatively mature neurons, but this structure develops slower and forms with a lower propensity in dendrites than in axons. In addition, we observed that spectrin, actin, and adducin also form a 2D polygonal lattice structure, resembling the expanded erythrocyte membrane skeleton structure, in the somatodendritic compartment. This 2D lattice structure also develops substantially more slowly in the soma and dendrites than the development of the 1D MPS in axons. These results suggest membrane skeleton structures are differentially regulated across different subcompartments of neurons.

Background All known biological functions of the pro-inflammatory cytokine interleukin-1β (IL-1β) are mediated by type 1 interleukin receptor (IL-1R1). IL-1β–IL-1R1 signaling modulates various neuronal functions including spinal pain processing. Although the role of IL-1β in pain processing is generally accepted, there is a discussion in the literature whether IL-1β exerts its effect on spinal pain processing by activating neuronal or glial IL-1R1. To contribute to this debate, here we investigated the expression and cellular distribution of IL-1R1 in the superficial spinal dorsal horn in control animals and also in inflammatory pain. Methods Experiments were performed on rats and wild type as well as IL-1R1-deficient mice. Inflammatory pain was evoked by unilateral intraplantar injection of complete Freund adjuvant (CFA). The nociceptive responsiveness of control and CFA-treated animals were tested daily for withdrawal responses to mechanical and thermal stimuli before and after CFA injection. Changes in the expression of 48 selected genes/mRNAs and in the quantity of IL-1R1 protein during the first 3 days after CFA injection were measured with the TaqMan low-density array method and Western blot analysis, respectively. The cellular localization of IL-1R1 protein was investigated with single and double staining immunocytochemical methods. Results We found a six times and two times increase in IL-1R1 mRNA and protein levels, respectively, in the dorsal horn of CFA-injected animals 3 days after CFA injection, at the time of the summit of mechanical and thermal allodynia. Studying the cellular distribution of IL-1R1, we found an abundant expression of IL-1R1 on the somatodendritic compartment of neurons and an enrichment of the receptor in the postsynaptic membranes of some excitatory synapses. In contrast to the robust neuronal localization, we observed only a moderate expression of IL-1R1 on astrocytes and a negligible one on microglial cells. CFA injection into the hind paw caused a remarkable increase in the expression of IL-1R1 in neurons, but did not alter the glial expression of the receptor. Conclusion The results suggest that IL-1β exerts its effect on spinal pain processing primarily through neuronal IL-1R1, but it can also interact in some extent with IL-1R1 expressed by astrocytes.

Neuronal chloride levels are developmentally regulated. Early in life, high intracellular concentrations support chloride efflux and depolarization at GABAergic synapses. In mouse, intracellular chloride decreases over the first postnatal week in the somatodendritic compartment, eventually supporting mature, hyperpolarizing GABAergic inhibition. In contrast to this dendritic switch, it is less clear how GABAergic signaling at the axon initial segment (AIS) functions in mature pyramidal cells, as reports of both depolarization and hyperpolarization have been reported in the AIS past the first postnatal week. Here, we show that GABAergic signaling at the AIS of prefrontal pyramidal cells, indeed, switches polarity from depolarizing to hyperpolarizing but does so over a protracted periadolescent period. This is the most delayed maturation in chloride reversal in any structure studied to date and suggests that chandelier cells, which mediate axo-axonic inhibition, play a unique role in the periadolescent maturation of prefrontal circuits.

High-frequency stimulation of the medial perforant path triggers robust phosphorylation of ribosomal protein S6 (rpS6) in activated dendritic domains and granule cell bodies. Here we dissect the signaling pathways responsible for synaptically driven rpS6 phosphorylation in the dentate gyrus using pharmacological agents to inhibit PI3-kinase/mTOR and MAPK/ERK-dependent kinases. Using phospho-specific antibodies for rpS6 at different sites (ser235/236 versus ser240/244), we show that delivery of the PI3-kinase inhibitor, wortmannin, decreased rpS6 phosphorylation throughout the somatodendritic compartment (granule cell layer, inner molecular layer, outer molecular layer), especially in granule cell bodies while sparing phosphorylation at activated synapses (middle molecular layer). In contrast, delivery of U0126, an MEK inhibitor, attenuated rpS6 phosphorylation specifically in the dendritic laminae leaving phosphorylation in the granule cell bodies intact. Delivery of the mTOR inhibitor, rapamycin, abolished activation of rpS6 phosphorylation in granule cell bodies and dendrites, whereas delivery of a selective S6K1 inhibitor, PF4708671, or RSK inhibitor, SL0101-1, attenuated rpS6 phosphorylation throughout the postsynaptic cell. These results reveal that MAPK/ERK-dependent signaling is predominately responsible for the selective induction of rpS6 phosphorylation at active synapses. In contrast, PI3-kinase/mTOR-dependent signaling induces rpS6 phosphorylation throughout the somatodendritic compartment but plays a minimal role at active synapses. Collectively, these results suggest a potential mechanism by which PI3-kinase/mTOR and MAPK/ERK pathways regulate translation at specific subcellular compartments in response to synaptic activity.

In highly polarized cells such as neurons, most RNA molecules are not randomly distributed but sorted into different compartments. So far, methods to analyze the transcriptome in distinct subcellular compartments are not well established. Here, we first describe the culturing of primary motoneurons in compartmentalized chambers to separate the axons from the somatodendritic compartment. Second, we introduce a method for whole transcriptome amplification followed by high-throughput sequencing to analyze the RNA composition of these two different compartments in neuronal cells.