Broader Insights into Understanding Tumor Necrosis Factor and Neurodegenerative Disease Pathogenesis Infer New Therapeutic Approaches.

Proinflammatory cytokines such as tumor necrosis factor (TNF), with its now appreciated key roles in neurophysiology as well as neuropathophysiology, are sufficiently well-documented to be useful tools for enquiry into the natural history of neurodegenerative diseases. We review the broader literature on TNF to rationalize why abruptly-acquired neurodegenerative states do not exhibit the remorseless clinical progression seen in those states with gradual onsets. We propose that the three typically non-worsening neurodegenerative syndromes, post-stroke, post-traumatic brain injury (TBI), and post cardiac arrest, usually become and remain static because of excess cerebral TNF induced by the initial dramatic peak keeping microglia chronically activated through an autocrine loop of microglial activation through excess cerebral TNF. The existence of this autocrine loop rationalizes post-damage repair with perispinal etanercept and proposes a treatment for cerebral aspects of COVID-19 chronicity. Another insufficiently considered aspect of cerebral proinflammatory cytokines is the fitness of the endogenous cerebral anti-TNF system provided by norepinephrine (NE), generated and distributed throughout the brain from the locus coeruleus (LC). We propose that an intact LC, and therefore an intact NE-mediated endogenous anti-cerebral TNF system, plus the DAMP (damage or danger-associated molecular pattern) input having diminished, is what allows post-stroke, post-TBI, and post cardiac arrest patients a strong long-term survival advantage over Alzheimer's disease and Parkinson's disease sufferers. In contrast, Alzheimer's disease and Parkinson's disease patients remorselessly worsen, being handicapped by sustained, accumulating, DAMP and PAMP (pathogen-associated molecular patterns) input, as well as loss of the LC-origin, NE-mediated, endogenous anti-cerebral TNF system. Adrenergic receptor agonists may counter this.

Neurodegenerative disease treatments by direct TNF reduction, SB623 cells, maraviroc and irisin and MCC950, from an inflammatory perspective - a Commentary.

Introduction: The importance of excessive cerebral tumor necrosis factor (TNF) concentrations as one of the central tenets of the pathogenesis of the neurodegenerative diseases is now widely known, but variably accepted. Areas covered: Here we update the field by including material that is freely available on the large databases, particularly PubMed. We include the therapeutic outcomes with etanercept (a widely used specific anti-TNF biological), XPro1595 (a new double negative TNF inhibitor), 3,61-dithiothalidomide, implanted SB623 stem cells, maraviroc (a CCR5 inhibitor used to treat AIDS), MCC950 (an NLRP3 inhibitor), and changes in the hormone irisin. Expert opinion: Remarkably, considering the ample literature that links SB623 cells, maraviroc, MCC950 and irisin to TNF, these publications do not mention this cytokine, and therefore not their implicit involvement with controlling its cerebral levels. With regard to developments demonstrated by MCC950, we note that DAMPs and PAMPs recognize and activate both TLRs and inflammasomes in these disease states. Here, as in other illnesses, data suggests that preventing a pathogenic interaction could be achieved through shutting down either of these arms of innate immunity.

Therapeutic implications of how TNF links apolipoprotein E, phosphorylated tau, α-synuclein, amyloid-ß and insulin resistance in neurodegenerative diseases.

While cytokines such as TNF have long been recognized as essential to normal cerebral physiology, the implications of their chronic excessive production within the brain are now also increasingly appreciated. Syndromes as diverse as malaria and lead poisoning, as well as non-infectious neurodegenerative diseases, illustrate this. These cytokines also orchestrate changes in tau, α-synuclein, amyloid-ß levels and degree of insulin resistance in most neurodegenerative states. New data on the effects of salbutamol, an indirect anti-TNF agent, on α-synuclein and Parkinson's disease, APOE4 and tau add considerably to the rationale of the anti-TNF approach to understanding, and treating, these diseases. Therapeutic advances being tested, and arguably useful for a number of the neurodegenerative diseases, include a reduction of excess cerebral TNF, whether directly, with a specific anti-TNF biological agent such as etanercept via Batson's plexus, or indirectly via surgically implanting stem cells. Inhaled salbutamol also warrants investigating further across the neurodegenerative disease spectrum. It is now timely to integrate this range of new information across the neurodegenerative disease spectrum, rather than keep seeing it through the lens of individual disease states.

Amyloid ß: one of three danger-associated molecules that are secondary inducers of the proinflammatory cytokines that mediate Alzheimer's disease.

This review concerns how the primary inflammation preceding the generation of certain key damage-associated molecular patterns (DAMPs) arises in Alzheimer's disease (AD). In doing so, it places soluble amyloid ß (Aß), a protein hitherto considered as a primary initiator of AD, in a novel perspective. We note here that increased soluble Aß is one of the proinflammatory cytokine-induced DAMPs recognized by at least one of the toll-like receptors on and in various cell types. Moreover, Aß is best regarded as belonging to a class of DAMPs, as do the S100 proteins and HMBG1, that further exacerbate production of these same proinflammatory cytokines, which are already enhanced, and induces them further. Moreover, variation in levels of other DAMPs of this same class in AD may explain why normal elderly patients can exhibit high Aß plaque levels, and why removing Aß or its plaque does not retard disease progression. It may also explain why mouse transgenic models, having been designed to generate high Aß, can be treated successfully by this approach.

Selective knockdown of NMDA receptors in primary afferent neurons decreases pain during phase 2 of the formalin test.

The role of NMDA receptors (NMDARs) expressed by primary afferent neurons in nociception remains controversial. The aim of this study was to develop mice with a tissue selective knockdown of NMDARs in these neurons and to evaluate their behavioral responses to different types of painful stimuli. Mice with floxed NMDAR NR1 subunit gene (fNR1) were crossed with mice expressing Cre recombinase under the control of the peripherin promotor (Prph-Cre). Male Prph-Cre+ floxed NR1 mice were compared to Cre- littermates. Both quantitative RT/PCR and Western blotting indicated a ∼75% reduction in NR1 expression in dorsal root ganglia (DRG) extracts with no effect on NR1 expression in spinal cord, brain or the enteric nervous system. Immunocytochemistry with antibodies to NR1 revealed decreased staining in all size classes of DRG neurons. NMDA produced a detectable increase in [Ca2+]i in 60% of DRG neurons cultured from Cre- mice, but only 15% of those from Cre+ mice. Furthermore, the peak [Ca2+]i responses were 64% lower in neurons from Cre+ mice. There was no significant difference between Cre+ and Cre- mice in response latencies to the hotplate or tail withdrawal tests of thermal nociception, nor was there a difference in withdrawal thresholds to mechanical stimuli of the tail or paw. However, compared to Cre- littermates, Cre+ knockdown mice had a 50% decrease in the phase 2 response to formalin injection (P<0.001). There was no effect on phase 1 responses. These results suggest that NMDA receptors expressed by primary afferent nerves play an important role in the development of sensitized pain states.

Functional expression of distinct NMDA channel subunits tagged with green fluorescent protein in hippocampal neurons in culture.

We generated expression vectors for N-terminally green fluorescent protein -tagged NR2A and NR2B subunits (GFP-NR2A and GFP-NR2B). Both constructs expressed GFP and formed functional NMDA channels with similar properties to untagged controls when co-transfected with NR1 subunit partner in HEK293 cells. Primary cultured hippocampal neurons were transfected at five days in vitro with these vectors. Fifteen days after transfection, well-defined GFP clusters were observed for both GFP-NR2A and GFP-NR2B subunits being co-localized with endogenous NR1 subunit. Whole-cell recordings showed that the GFP-NR2A subunit determined the decay of NMDA-mediated miniature spontaneous excitatory postsynaptic currents (NMDA-mEPSCs) in transfected neurons. Live staining with anti-GFP antibody demonstrated the surface expression of GFP-NR2A and GFP-NR2B subunits that was partly co-localized a presynaptic marker. Localization of NMDA receptor clusters in dendrites was studied by co-transfection of CFP-actin and GFP-NR2 subunits followed by anti-GFP surface staining. Within one week after plating most surface NMDAR clusters were distributed on dendritic shafts. Later in development, a large portion of surface clusters for both GFP-NR2A and GFP-NR2B subunits were clearly localized at dendritic spines. Our report provides the basis for studies of NMDA receptor location together with dendritic dynamics in living neurons during synaptogenesis in vitro.

A use-dependent tyrosine dephosphorylation of NMDA receptors is independent of ion flux.

Tyrosine phosphorylation can upregulate NMDA receptor activity during pathological and physiological alterations of synaptic strength. Here we describe downregulation of recombinant NR1/2A receptors by tyrosine dephosphorylation that requires agonist binding, but is independent of ion flux. The tyrosine residues involved in this new form of NMDA receptor modulation likely form a 'ring' adjacent to the last transmembrane domain. The downregulation was due to a reduction in the number of functional channels, and was blocked by co-expressing a dominant-negative mu2-subunit of the clathrin-adaptor protein AP-2. Our results provide a mechanism by which synaptic NMDA receptors can be modulated in a use-dependent manner even when the postsynaptic membrane is not sufficiently depolarized to relieve channel block by magnesium ions.

The role of RNA editing of kainate receptors in synaptic plasticity and seizures.

The ionotropic glutamate receptor subunit GluR6 undergoes developmentally and regionally regulated Q/R site RNA editing that reduces the calcium permeability of GluR6-containing kainate receptors. To investigate the functional significance of this editing in vivo, we engineered mice deficient in GluR6 Q/R site editing. In these mutant mice but not in wild types, NMDA receptor-independent long-term potentiation (LTP) could be induced at the medial perforant path-dentate gyrus synapse. This indicates that kainate receptors with unedited GluR6 subunits can mediate LTP. Behavioral analyses revealed no differences from wild types, but mutant mice were more vulnerable to kainate-induced seizures. Together, these results suggest that GluR6 Q/R site RNA editing may modulate synaptic plasticity and seizure vulnerability.

Interactions of calmodulin and alpha-actinin with the NR1 subunit modulate Ca2+-dependent inactivation of NMDA receptors.

Glutamate receptors are associated with various regulatory and cytoskeletal proteins. However, an understanding of the functional significance of these interactions is still rudimentary. Studies in hippocampal neurons suggest that such interactions may be involved in calcium-induced reduction in the open probability of NMDA receptors (inactivation). Thus we examined the role of the intracellular domains of the NR1 subunit and two of its binding partners, calmodulin and alpha-actinin, on this process using NR1/NR2A heteromers expressed in human embryonic kidney (HEK) 293 cells. The presence of the first 30 residues of the intracellular C terminus of NR1 (C0 domain) was required for inactivation. Mutations in the last five residues of C0 reduced inactivation and produced parallel shifts in binding of alpha-actinin and Ca2+/calmodulin to the respective C0-derived peptides. Although calmodulin reduced channel activity in excised patches, calmodulin inhibitors did not block inactivation in whole-cell recording, suggesting that inactivation in the intact cell is more complex than binding of calmodulin to C0. Overexpression of putative Ca2+-insensitive, but not Ca2+-sensitive, forms of alpha-actinin reduced inactivation, an effect that was overcome by inclusion of calmodulin in the whole-cell pipette. The C0 domain also directly affects channel gating because NR1 subunits with truncated C0 domains that lacked calmodulin or alpha-actinin binding sites had a low open probability. We propose that inactivation can occur after C0 dissociates from alpha-actinin by two distinct but converging calcium-dependent processes: competitive displacement of alpha-actinin by calmodulin and reduction in the affinity of alpha-actinin for C0 after binding of calcium to alpha-actinin.

N-terminal domains in the NR2 subunit control desensitization of NMDA receptors.

Recent molecular studies of glutamate channels have provided increasingly detailed models of the agonist-binding site and of the channel pore. However, little information is available on the domains involved in channel gating. We examined the molecular determinants for the NR2-subunit specificity of glycine-independent desensitization of NMDA channels using NR2C/NR2A chimeric subunits expressed in HEK 293 cells. We show that glycine-independent desensitization is controlled by N-terminal domains of the NR2 subunit that flank the putative agonist-binding domain: a four amino acid (aa) segment immediately preceding the first transmembrane domain (M1) and a region containing the leucine/isoleucine/valine-binding protein-like (LIVBP-like) domain. Our results provide evidence for a functional role of the region containing the LIVBP-like domain in glutamate receptor channels. We suggest that the pre-M1 segment, presumably situated near the entrance to the pore, serves as a dynamic link between ligand binding and channel gating.

Cytoskeletal interactions with glutamate receptors at central synapses.

The data presented here are clearly just the beginning of any comprehensive understanding of the set of regulatory and cytoskeletal proteins that interact with membrane receptors in the postsynaptic density. They do, however, indicate that both glutamate channels at central excitatory synapses are involved in complex protein-protein interactions. For example, while NR2A is important for Ca-dependent inactivation of NMDA receptors, studies in several systems suggest that the other major NR2 subunit in hippocampal neurons, NR2B, predominates at critical times during synapse formation. In addition, the COOH terminus of NR2B binds to several novel cytoskeletal proteins. These results provide circumstantial evidence that NR2B may play specific roles in function and localization of receptors at excitatory synapses. The possible role of NR2B in early synaptic function gains additional support from functional data suggesting that NMDA receptors have specific roles during development (Komuro and Rakic, 1993; Rabacchi et al., 1992; Yen et al., 1993). The essential role of NR1 and NR2B in development is graphically demonstrated by the neonatal death of transgenic mice lacking either of these two subunits (Forrest et al., 1994; Kutsuwada et al., 1996) whereas NR2A and NR2C-deficient mice are less severely affected (Sakimura et al., 1995; Ebralidze et al., 1996).

Calcium-dependent inactivation of recombinant N-methyl-D-aspartate receptors is NR2 subunit specific.

Intracellular Ca2+ can reversibly reduce the activity of native N-methyl-D-aspartate (NMDA) receptors in hippocampal neurons, a phenomenon termed Ca2+-dependent inactivation. We examined inactivation in heteromeric NMDA receptors expressed in human embryonic kidney (HEK) 293 cells using whole-cell recording. NR1-1a/2A heteromers showed reversible inactivation that was very similar to native NMDA receptors in cultured hippocampal neurons. Inactivation was dependent on the extracellular Ca2+ concentration and the degree of intracellular Ca2+ buffering. In 2 mM extracellular Ca2+, inactivation resulted in a 46.1 +/- 12.6% reduction in the whole-cell current during a 5-sec agonist application. Inactivation of NR1-1a/2A heteromers was unaffected by calcineurin inhibitors, staurosporine, or phalloidin. NR1-1a/2D heteromers also showed a similar degree of inactivation. In contrast, NR1-1a/2B and NR1-1a/2C heteromers showed no significant inactivation. At saturating concentrations of NMDA (1 mM), NR1-1a/2A heteromers also showed Ca- and glycine-independent desensitization, as seen in native hippocampal neurons. Ca(2+)- and glycine-independent desensitization was less pronounced in NR1-1a/2B heteromers and absent in NR1-1a/2C heteromers. Activation of NR1-1a/2C heteromers triggered intracellular Ca2+ transients similar to NR1-1a/2A heteromers as verified by combined Ca2+ imaging and whole-cell recording. Thus differences in Ca2+ permeability were not responsible for the lack of inactivation in NR1-1a/2C heteromers. Our results show that inactivation of recombinant NMDA receptors requires either the NR2A or NR2D subunit, whereas both inactivation and desensitization were absent in NR2C-containing receptors. The gating of inactivating NMDA receptors is more likely to be influenced by ongoing NMDA receptor activity and Ca2+ transients, perhaps consistent with the prominent expression of NR2A in hippocampus and cerebral cortex.

Long-range analyses of the centromeric regions of human chromosomes 13, 14 and 21: identification of a narrow domain containing two key centromeric DNA elements.

Alpha-satellite, satellite 3 and satellite 1 DNA have been proposed as candidate components of a functional human centromere. Multiple subfamilies of these three DNA have recently been identified at the pericentric regions of the human acrocentric chromosomes. Using pulsed field gel electrophoresis, we have constructed long-range maps of the various centromeric markers for chromosomes 13, 14 and 21. These maps cover approximately 2.3 megabases of sequence for each chromosome, and the results demonstrate that within this centromeric region, chromosomes 13 and 21 have a similar organization that is partially shared by chromosome 14. A discrete satellite 3 domain was identified on each chromosome within the boundaries of the alpha-satellite DNA. No satellite 1 was detected within the defined centromeric regions suggesting that satellite 1 is not essential for centromere function.

A chromosome 13-specific human satellite I DNA subfamily with minor presence on chromosome 21: further studies on Robertsonian translocations.

We describe a new human satellite I DNA subfamily (pTRI-6) which is composed of 72 copies of monomeric repeating units of 42 basepairs (bp). These repeating units are tandemly organized into a higher order structure of 2.97 kilobases (kb). Sequencing of this DNA revealed base substitutions, deletions and insertions, and an overall conservation of 85% among the monomers. The sequence has a high AT content of 77%. Under low-stringency in situ hybridization conditions, satellite I is found on the pericentric regions of chromosomes 3 and 4 and on all the acrocentric chromosomes. On the acrocentric chromosomes, satellite I is further detected on the distal p13 region. Analysis of somatic cell hybrids under high stringency indicates the presence of the pTRI-6 subfamily predominantly on chromosome 13. Chromosome 21 shows a 50- to 100-fold reduced amount of this subfamily and the presence of other sequences closely related to pTRI-6. Investigation of a group of 11 human t(14q21q) Robertsonian translocations revealed the retention of satellite I DNA around the breakpoints in all cases. These results extend our understanding of these translocations and of the general structural organization of the cen-pter regions of the acrocentric chromosomes.

Evolutionary relationships of multiple alpha satellite subfamilies in the centromeres of human chromosomes 13, 14, and 21.

Using Southern and in situ hybridization analyses, we have earlier defined four different subfamilies of alpha satellite DNA (designated pTRA-1, -2, -4, and -7), each of which has a unique higher order structure represented almost identically on human chromosomes 13, 14, and 21. Here we present the complete sequence of single isolates of these four subfamilies, representing approximately 12 kb of sequence information. Sequences of the individual 171-bp monomers that constitute these four subfamilies (and a fifth subfamily, Alpha-R1, that is known to be present on chromosomes 13 and 21) were compared both within and between the different clones. The results indicate that, at the level of their primary sequence, the five alpha subfamilies are characterized by structures that are as unrelated to each other as the different alpha subfamilies from other chromosomes. However, sequence comparisons between monomers of these clones indicate the possibility that pTRA-2, -4, and -1 may have arisen, at least in part, from a common ancestral alphoid sequence. We also provide evidence that exchange of pTRA-1 between nonhomologous centromeres and its homogenization throughout the population, perhaps by unequal exchange mechanisms, could have occurred after the divergence of humans and chimpanzees. The evolution of multiple alphoid subfamilies within a single centromere suggests that unequal exchange mechanisms may be restricted to specific domains. This may in turn contribute to some requirement for subregional pairing of sequences along the length of the centromeres of these chromosomes.

A chromosome 14-specific human satellite III DNA subfamily that shows variable presence on different chromosomes 14.

We describe a new subfamily of satellite III DNA (pTRS-63), which, by a combination of in situ hybridization to human metaphase chromosomes and analysis of a panel of somatic cell hybrids, is shown to be specific for human chromosome 14. This DNA has a basic 5-bp repeating unit of diverged GGAAT which is tandemly repeated and organized into either one of two distinct higher-order structures of 5 kb (designated the "L" form) or 4.8 kb (designated the "S" form). In addition, a third (Z) form, representing no detectable levels of this satellite III subfamily, is found. Results from five somatic cell hybrid lines and from a number of informative human individuals suggest that, on any one chromosome 14, only one of the three forms may exist. Subchromosomally, this sequence has been mapped to the p11 region and is distal to the domain occupied by another previously described satellite III subfamily (pTRS-47) found on chromosome 14. The pTRS-63 sequence described adds to the understanding of the structural organization of the short arm of human chromosome 14 and should be useful for the investigation of the molecular etiology of the frequently occurring t(13q14q) and t(14q21q) Robertsonian translocations.

A satellite III sequence shared by human chromosomes 13, 14, and 21 that is contiguous with alpha satellite DNA.

We report the isolation of a clone (pTR9) from a human chromosome 21 lambda phage library, which was found to contain two distinct components: (1) a previously unreported subfamily of human satellite III (pTR9-s3; 1,485 bp) and (2) an alpha satellite sequence (pTR9-alpha; 250 bp) containing 1.5 copies of a 171-bp alphoid unit that shows 88.4% homology to a previously reported alpha satellite consensus sequence. The two components are separated by two direct repeats of 9 bp. Use of the polymerase chain reaction (PCR) to amplify across the junction between pTR9-s3 and pTR9-alpha established that these two sequences are contiguous in total human genomic DNA and in DNA derived from somatic cell hybrids carrying human chromosomes 13, 14, or 21. A related, but considerably more diverged, sequence was also detected on chromosome 15. Southern analysis of somatic cell hybrids at high stringency revealed a common structure of the pTR9-s3 sequence on chromosomes 13, 14, and 21 but not on 15 or 22. This sequence should be useful for the study of the structural organisation of the centromere of these chromosomes and the mechanism of their involvement in Robertsonian translocations.

Four distinct alpha satellite subfamilies shared by human chromosomes 13, 14 and 21.

We describe the characterisation of four alpha satellite sequences which are found on a subset of the human acrocentric chromosomes. Direct sequence study, and analysis of somatic cell hybrids carrying specific human chromosomes indicate a unique 'higher-order structure' for each of the four sequences, suggesting that they belong to different subfamilies of alpha DNA. Under very high stringency of Southern hybridisation conditions, all four subfamilies were detected on chromosomes 13, 14 and 21, with 13 and 21 showing a slightly greater sequence homology in comparison to chromosome 14. None of these subfamilies were detected on chromosomes 15 and 22. In addition, we report preliminary evidence for a new alphoid subfamily that is specific for human chromosome 14. These results, together with those of earlier published work, indicate that the centromeres of the five acrocentric chromosomes are characterised by a number of clearly defined alphoid subfamilies or microdomains (with at least 5, 7, 3, 5 and 2 different ones on chromosomes 13, 14, 15, 21 and 22, respectively). These microdomains must impose a relatively stringent subregional pairing of the centromeres of two homologous chromosomes. The different alphoid subfamilies reported should serve as useful markers to allow further 'dissection' of the structure of the human centromere as well as the investigation of how the different nonhomologous chromosomes may interact in the aetiology of aberrations involving these chromosomes.

Identification of two distinct subfamilies of alpha satellite DNA that are highly specific for human chromosome 15.

We report the isolation of two distinct subfamilies of alpha satellite DNA (pTRA-20 and -25) from human chromosome 15. In situ hybridization experiments indicated that both subfamilies are highly specific for this chromosome. Southern analysis of a somatic hybrid cell line carrying human chromosome 15 revealed a likely higher-order genomic band of 2.5 kb for pTRA-20. Similar analysis for pTRA-25 showed multiple higher-order bands of 3.5, 4.5, and 5 kb at moderately high hybridization stringency, but a predominance of the 4.5-kb species at very high stringency. Direct comparison with human genomic DNA confirmed the authenticity of these higher-order structures and demonstrated polymorphic variations using both probes. The origin of the different alphoid subfamilies on chromosome 15 is discussed. These sequences should be useful for the construction of centromere-based genetic linkage maps for human chromosome 15 and, in conjunction with the other alphoid sequences already reported for chromosomes 13, 14, 21, and 22, should allow a concerted analysis of the evolution and the possible etiological role of these DNAs in aberrations commonly seen in these chromosomes.