Journal of Neurochemistry最新文献

As one of the most important cellular housekeepers, autophagy directly affects cellular health, homeostasis, and function. Even though the mechanisms behind autophagy are well described, how molecular alterations and dysfunctions can lead to pathology in disease contexts still demands deeper investigation. Proteomics is a widely employed tool used to investigate molecular alterations associated with pathological states and has proven useful in identifying alterations in protein expression levels and post-translational modifications in autophagy. In this narrative review, we expand on the molecular mechanisms behind autophagy and its regulation, and further compile recent literature associating autophagy disturbances in context of brain disorders, utilizing discoveries from varying models and species from rodents and cellular models to human post-mortem brain samples. To outline, the canonical pathways of autophagy, the effects of post-translational modifications on regulating each step of autophagy, and the future directions of proteomics in autophagy will be discussed. We further aim to suggest how advancing proteomics can help further unveil molecular mechanisms with regard to neurological disorders.

Myelin is a vital structure that is key to rapid saltatory conduction in the central and peripheral nervous systems. Much work has been done over the decades examining the biochemical composition and morphology of myelin at the light and electron microscopic levels. Here we report a method to study myelin based on the fluorescent probe Nile Red. This lipophilic dye readily partitions into live and chemicallyfixed myelin producing bright, well-resolved images of the sheath. Using spectral confocal microscopy, a complete emission spectrum of Nile Red fluorescence can be acquired for each pixel in an image. The solvatochromic properties of Nile Red cause its emission spectrum to change depending on the polarity of its local environment. Therefore, measuring spectral shifts can report subtle changes in the physicochemical properties of myelin. We show differences in myelin polarity in central versus peripheral nervous system and in different regions of central nervous system white matter of the mouse brain, together with developmental and sex variations. This technique is also well suited for measuring subtle changes in myelin properties in live ex vivo white matter specimens. We also demonstrate how light deprivation induces a myelin polarity change in adult mouse optic nerve underscoring a continuing myelin plasticity in response to axonal activity well into adulthood. The Nile Red spectroscopic method allows measurement of subtle physicochemical changes in myelin that can importantly influence its electrical properties and by extension, conduction velocities in axons.

Myelin is an insulator that forms around axons that enhance the conduction velocity of nerve fibers. Oligodendrocytes dramatically change cell morphology to produce myelin throughout the central nervous system (CNS). Cytoskeletal alterations are critical for the morphogenesis of oligodendrocytes, and actin is involved in cell differentiation and myelin wrapping via polymerization and depolymerization, respectively. Various protein members of the myosin superfamily are known to be major binding partners of actin filaments and have been intensively researched because of their involvement in various cellular functions, including differentiation, cell movement, membrane trafficking, organelle transport, signal transduction, and morphogenesis. Some members of the myosin superfamily have been found to play important roles in the differentiation of oligodendrocytes and in CNS myelination. Interestingly, each member of the myosin superfamily expressed in oligodendrocyte lineage cells also shows specific spatial and temporal expression patterns and different distributions. In this review, we summarize previous findings related to the myosin superfamily and discuss how these molecules contribute to myelin formation and regeneration by oligodendrocytes.

The amyloid β (Aβ) peptide has a central role in Alzheimer's disease (AD) pathology. The peptide length can vary between 37 and 49 amino acids, with Aβ1-42 being considered the most disease-related length. However, Aβ1-40 is also found in Aβ plaques and has shown to form intertwined fibrils with Aβ1-42. The peptides have previously also shown to form different fibril conformations, proposed to be related to disease phenotype. To conduct more representative in vitro experiments, it is vital to uncover the impact of different fibril conformations on neurons. Hence, we fibrillized different Aβ1-40:42 ratios in concentrations of 100:0, 90:10, 75:25, 50:50, 25:75, 10:90 and 0:100 for either 24 h (early fibrils) or 7 days (aged fibrils). These were then characterized based on fibril width, LCO-staining and antibody-staining. We further challenged differentiated neuronal-like SH-SY5Y human cells with the different fibrils and measured Aβ content, cytotoxicity and autophagy function at three different time-points: 3, 24, and 72 h. Our results revealed that both Aβ1-40:42 ratio and fibril maturation affect conformation of fibrils. We further show the impact of these conformation changes on the affinity to commonly used Aβ antibodies, primarily affecting Aβ1-40 rich aggregates. In addition, we demonstrate uptake of the aggregates by neuronally differentiated human cells, where aggregates with higher Aβ1-42 ratios generally caused higher cellular levels of Aβ. These differences in Aβ abundance did not cause changes in cytotoxicity nor in autophagy activation. Our results show the importance to consider conformational differences of Aβ fibrils, as this can have fundamental impact on Aβ antibody detection. Overall, these insights underline the need for further exploration of the impact of conformationally different fibrils and the need to reliably produce disease relevant Aβ aggregates.

The strongest genetic risk factor for late-onset Alzheimer's disease (AD) is allelic variation of the APOE gene, with the following risk structure: ε4 > ε3 > ε2. The biochemical basis for this risk profile is unclear. Here, we reveal a new role for the APOE gene product, apolipoprotein E (ApoE) in regulating cellular copper homeostasis, which is perturbed in the AD brain. Exposure of ApoE target replacement (TR) astrocytes (immortalised astrocytes from APOE knock-in mice) to elevated copper concentrations resulted in exacerbated copper accumulation in ApoE4- compared to ApoE2- and ApoE3-TR astrocytes. This effect was also observed in SH-SY5Y neuroblastoma cells treated with conditioned medium from ApoE4-TR astrocytes. Increased intracellular copper levels in the presence of ApoE4 may be explained by reduced levels and delayed trafficking of the copper transport protein, copper-transporting ATPase 1 (ATP7A/Atp7a), potentially leading to impaired cellular copper export. This new role for ApoE in copper regulation lends further biochemical insight into how APOE genotype confers risk for AD and reveals a potential contribution of ApoE4 to the copper dysregulation that is a characteristic pathological feature of the AD brain.

Dynamins are large GTPases whose primary function is not only to catalyze membrane scission during endocytosis but also to modulate other cellular processes, such as actin polymerization and vesicle trafficking. Recently, we reported that centronuclear myopathy associated dynamin-2 mutations, p.A618T, and p.S619L, impair Ca²⁺-induced exocytosis of the glucose transporter GLUT4 containing vesicles in immortalized human myoblasts. As exocytosis and endocytosis occur within rapid timescales, here we applied high-temporal resolution techniques, such as patch-clamp capacitance measurements and carbon-fiber amperometry to assess the effects of these mutations on these two cellular processes, using bovine chromaffin cells as a study model. We found that the expression of any of these dynamin-2 mutants inhibits a dynamin and F-actin-dependent form of fast endocytosis triggered by single action potential stimulus, as well as inhibits a slow compensatory endocytosis induced by 500 ms square depolarization. Both dynamin-2 mutants further reduced the exocytosis induced by 500 ms depolarizations, and the frequency of release events and the recruitment of neuropeptide Y (NPY)-labeled vesicles to the cell cortex after stimulation of nicotinic acetylcholine receptors with 1,1-dimethyl-4-phenyl piperazine iodide (DMPP). They also provoked a significant decrease in the Ca²⁺-induced formation of new actin filaments in permeabilized chromaffin cells. In summary, our results indicate that the centronuclear myopathy (CNM)-linked p.A618T and p.S619L mutations in dynamin-2 affect exocytosis and endocytosis, being the disruption of F-actin dynamics a possible explanation for these results. These impaired cellular processes might underlie the pathogenic mechanisms associated with these mutations.

The impact of primary and secondary injuries of spinal cord injury (SCI) results in the demise of numerous neurons, and there is still no efficacious pharmacological intervention for it. Recently, studies have shown that endoplasmic reticulum stress (ERS) plays a pivotal role in recovery of neurological function after spinal cord injury. As a process to cope with intracellular accumulation of misfolded and unfolded proteins which triggers ERS, the unfolded protein response (UPR) plays an important role in maintaining protein homeostasis. And, a recently disclosed small molecule AA147, which selectively activates activating transcription factor 6 (ATF6), has shown promising pharmacological effects in several disease models. Thus, it seems feasible to protect the neurons after spinal cord injury by modulating UPR. In this study, primary neurons were isolated from E17-19 C57BL/6J mouse embryos and we observed that AA147 effectively promoted the survival of neurons and alleviated neuronal apoptosis after oxygen–glucose deprivation/reoxygenation (OGD/R) in vitro. This was evident through a decrease in the proportion of PI-positive and TUNEL-positive cells, an increase in BCL-2 expression, and a decrease in the expression of BAX and C-caspase3. In in-vivo experiments, these findings were corroborated by TUNEL staining and immunohistochemistry. It was also found that AA147 enhanced three arms of the unfolded protein response with reduced CHOP expression. Besides, AA147 mitigated the accumulation of ROS in neurons probably by upregulating catalase expression. Furthermore, spinal cord injury models of C57BL/6J mice were established and behavioral experiments revealed that AA147 facilitated the recovery of motor function following SCI. Thus, pharmacologic activation of ATF6 represents a promise therapeutic approach to ameliorate the prognosis of SCI.

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system with a strong neuroinflammatory component. Current treatments principally target the immune system but fail to preserve long-term myelin health and do not prevent neurological decline. Studies over the past two decades have shown that the structurally related neuropeptides VIP and PACAP (vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide, respectively) exhibit pronounced anti-inflammatory activities and reduce clinical symptoms in MS disease models, largely via actions on their bivalent VIP receptor type 1 and 2. Here, using the cuprizone demyelination model, we demonstrate that PACAP and VIP, and strikingly the PACAP-selective receptor PAC1 agonist maxadilan, prevented locomotor deficits in the horizontal ladder and open field tests. Moreover, only PACAP and maxadilan were able to prevent myelin deterioration, as assessed by a reduction in the expression of the myelin markers proteolipid protein 1, oligodendrocyte transcription factor 2, quaking-7 (APC) and Luxol Fast Blue staining. Furthermore, PACAP and maxadilan (but not VIP), prevented striatal synaptic loss and diminished astrocyte and microglial activation in the corpus callosum of cuprizone-fed mice. In vitro, PACAP or maxadilan prevented lipopolysaccharide (LPS)-induced polarisation of primary astrocytes at 12–24 h, an effect that was not seen with maxadilan in LPS-stimulated microglia. Taken together, our data demonstrates for the first time that PAC1 agonists provide distinctive protective effects against white matter deterioration, neuroinflammation and consequent locomotor dysfunctions in the cuprizone model. The results indicate that targeting the PAC1 receptor may provide a path to treat myelin-related diseases in humans.

The nematode Caenorhabditis elegans is well known for its ability to support forward genetic screens to identify molecules involved in neuronal viability and signaling. The proteins involved in C. elegans dopamine (DA) regulation are highly conserved across evolution, with prior work demonstrating that the model can serve as an efficient platform to identify novel genes involved in disease-associated processes. To identify novel players in DA signaling, we took advantage of a recently developed library of pre-sequenced mutant nematodes arising from the million mutation project (MMP) to identify strains that display the DA-dependent swimming-induced-paralysis phenotype (Swip). Our screen identified novel mutations in the dopamine transporter encoding gene dat-1, whose loss was previously used to identify the Swip phenotype, as well as multiple genes with previously unknown connections to DA signaling. Here, we present our isolation and characterization of one of these genes, bbs-1, previously linked to the function of primary cilia in worms and higher organisms, including humans, and where loss-of-function mutations result in a human disorder known as Bardet–Biedl syndrome. Our studies of C. elegans BBS-1 protein, as well as other proteins that are known to be assembled into a higher order complex (the BBSome) reveal that functional or structural disruption of this complex leads to exaggerated C. elegans DA signaling to produce Swip via a cell-autonomous mechanism. We provide evidence that not only does the proper function of cilia in C. elegans DA neurons support normal swimming behavior, but also that bbs-1 maintains normal levels of DAT-1 trafficking or function via a RHO-1 and SWIP-13/MAPK-15 dependent pathway where mutants may contribute to Swip independent of altered ciliary function. Together, these studies demonstrate novel contributors to DA neuron function in the worm and demonstrate the utility and efficiency of forward genetic screens using the MMP library.

The accumulation of β-amyloid in Alzheimer's disease greatly impacts neuronal health and synaptic function. To maintain network stability in the face of altered synaptic activity, neurons engage a feedback mechanism termed homeostatic scaling; however, this process is thought to be disrupted during disease progression. Previous proteomics studies have shown that one of the most highly regulated proteins in cell culture models of homeostatic scaling is the small secretory chaperone proSAAS. Our prior work has shown that proSAAS exhibits anti-aggregant behavior against alpha-synuclein and β-amyloid fibrillation in vitro and is up-regulated in cell models of proteostatic stress. However, the specific role that this protein might play in homeostatic scaling, and its anti-aggregant role in Alzheimer's progression, is not clear. To learn more about the role of proSAAS in maintaining hippocampal proteostasis, we compared its expression in a primary neuron model of homeostatic scaling to other synaptic components using western blotting and qPCR, revealing that proSAAS protein responses to homeostatic up- and down-regulation were significantly higher than those of two other synaptic vesicle components, 7B2 and carboxypeptidase E. However, proSAAS mRNA expression was static, suggesting translational control and/or altered protein degradation. ProSAAS was readily released upon depolarization of differentiated hippocampal cultures, supporting its synaptic localization. Immunohistochemical analysis demonstrated abundant proSAAS within the mossy fiber layer of the hippocampus in both wild-type and 5xFAD mice; in the latter, proSAAS was also concentrated around amyloid plaques. Importantly, overexpression of proSAAS in the CA1 region via stereotaxic injection of proSAAS-encoding AAV2/1 significantly decreased amyloid plaque burden in 5xFAD mice. We hypothesize that dynamic changes in proSAAS expression play a critical role in hippocampal proteostatic processes, both in the context of normal homeostatic plasticity and in the control of protein aggregation during Alzheimer's disease progression.