A Platform for Parameter Unlimited Molecular Geometry Imaging Obviously Enabling Life Saving Measures in ALS

This article deals with a molecular geometry imaging platform capable of mapping the spatial protein-colocalisation and anti-colocalisation code of large molecular systems at a time. The platform called molecular unlimited systems imaging cycler (MUSIC) robotics was applied to amyotrophic lateral sclerosis (ALS). The detection of ALS specific cells with a corresponding multimolecular geometry in the blood led to therapeutic depletion of these cells and to recovery of the treated patient, obviously because this therapy interferes with pathogenic invasion of these cells into the central nervous system, where they axotomize motor axons. Large scale geometry MUSIC robotics imaging of up to 4.5 × 10 power of combinatorial molecular resolution is key to detect these cells and to control depletion therapy for clinical success. These data and new possibilities may argue for clinical application and for a systematic research in the field of molecular geometry of diseases to discover new mathematically defined insight.


Introduction
All life processes are spatial. Therefore, the molecular networks controlling life processes must also follow spatial laws. In order to understand these laws, methods must be developed that allow these networks to be imaged in a hypothe- sis-free manner, for an unlimited, or at least extremely large number of defined molecules and to directly distinguish the disease specific from the non specific networks in the morphologically intact tissue, optionally in real time. The development of the molecular unlimited systems imaging cycler (MUSIC) robotics method represents a fundamental technique that allows us to spatially map the so-called toponome, the spatial protein network, as a geometric data set with hundreds or thousands of interlinked combinatorial molecular clusters, to detect the resulting so called lead proteins or whole cells controlling these networks [1] [2]. These steps have proven to be fundamental when it comes to understanding their laws and eliminating disease-specific, so-called robustness networks, in a targeted therapeutic way [3], as shown here for ALS.
The question of whether these natural processes follow exact combination rules of proteins in health and disease has been answered in recent years by the imaging cycler robotics technology in healthy and diseased human tissues [1] [3] [4] [5] [6] [7]. According to this, protein systems in tissues can be understood as a system that follows a kind of spatial protein association, tissue-bound "grammar" and a protein association "vocabulary". To decode this system is therefore mandatory when it comes to understand diseases because of the following reasons: with only a limited number of rules for the spatial tissue association of e.g.
proteins, a quasi-unlimited number of spatial "texts" (diseases) with different contents (toponomes) can be expressed in tissues. These "texts" can, for example, be understood as spatial blueprints for disease, which can be decoded directly by means of imaging cycler robotics as a toponomical data set [7] [8] [9].
These spatial blueprints, in turn, typically include so-called emergent systems [10], where emergence is understood as the process of complex pattern formation from more basic constituent parts [10]: large combinatorial molecular structures acquire, e.g. through the differential interplay of the numerous different protein classes, new properties in health and disease [10].
To elucidate these characteristics as specific features of diseases appears to be the "conditio sine qua non" for the development of efficient treatments of chronic diseases, such as cancer or amyotrophic lateral sclerosis (ALS) [10]. This is supported by recent clinical success [11] based on toponome predicions [7] concerning the lead protein function of aminopeptidase N in cancer. So it's bound to be very erroneous to assume that from lists of extracted proteins (from tissue) disease-specific information can be gained. On the opposite: there must be a very specific spatial combinatorial order of proteins: this view is further supported by the fact that proteins in the Golgi apparatus of the cell are immediately sorted into specific, highly non random contexts with other proteins in vesicles which are then transported to their destination in the same cell and so on. Hence, there are plenty of biologically and anatomically motivated reasons speaking for a systematic decoding of large molecular systems in their natural structurally defined environment. Here the fundamentals of MUSIC robotics geometry mapping and the case of ALS are described as an example.

MUSIC Robotics Platform Bypassing the Spectral Resolution Limit
The detection of several different proteins in tissue sections or single cells, e.g.
the detection of 4 different proteins at a time, is possible in optical routine microscopy with the aid of fluorescence microscopy. But fluorochrome-specific wavelengths must be selected with the aid of filters, which limits the colocalization of different protein classes down to only 4 to 5 within the limited spectrum of visible light ranging from 300 to 700 nm wavelengths: too few separable parameters to determine the high-dimensional networks of proteins or other molecular classes to analyse and understand their higher order combinatorial laws.
However, due to the so-called sunlight hypothesis set up by the author of the present article in 1987 and described in [10], these limitations can be overcome or bypassed by using only one wavelength trace with sunlight-like soft scattered light to sequentially map many different molecules (100 or 1000 or more) with only one fluorochrome, if the fluorochrome is gently bleached before the next round for the next molecule is run. This so called sunlight hypothesis was experimentally verified in a two-page paper [12] and henceforth used for the development of an automated platform: the molecular unlimited systems imaging cycler robotics technology.

Direct Detection of Emergent Molecular Systems in Human Tissue
Two methods for the visualization of emergent molecular systems have been developed: the method of threshold-based analysis of molecular networks [7] [9] [12] [13] and the so-called similarity mapping method [14]. Both methods allow for analysis of high-dimensional molecular networks directly in the tissue on the basis of visualizing and quantifying a relative molecular stoichiometry of the different molecules ( Figure 1). The threshold-based method reveals so-called lead proteins that control large molecular networks ( Figure 1, top middle) [7] [8] (see also note added in proof). This provided evidence that the blocking of such lead proteins leads to a dramatic effect, by decomposing entire networks of proteins spatially [10]. For example, inhibition of the enzyme aminopeptidase N (APN), which controls a complete molecular network as a lead protein in rhabdomyosarcoma cells, results in the complete collapse of the molecular network, which normally enables cells to reach the elongated migration status [7]. Hence, in the meantime, this blocking method for this enzyme in cancers has been in positive validation as a therapeutic target in numerous clinical trials [11]. These data show that the MUSIC robotics method for detecting such relationships in disease-specific emergent systems is, for example, aimed at developing efficient therapeutic methods.

Emergent Molecular System in ALS
The This pathology was previously unknown because of a lack of combinatorial molecular visualization methods with emergence resolution, so that the pathogenesis of ALS was not resolvable at high functional resolution [3]. The conservative colocalisation of the two markers CD8 and CD3 (Figure 4(g)) is, analogous to   (a) to (k) is described in the text. In addition, the fact that the cell (c) inside the blood vessel is crossing the endothelial barrier (yellow) is given by the fact that its cell membrane (red) overlaps with the endothelial cell profile (yellow) and has already reached the nervous tissue as indicated by the arrow 2 in the image (c). During this transendothelial migration the cell extrudes vesicles containing CD16 and CD8, because these two lead proteins are not needed in this cell state any more: they were essential part of the cell state of rolling having preceded the cell state of transmigration. (g) After invasion the CD8 CD 3 positive cell is found in the parenchyma between myelinated motor nerves (g, blue and red dots). If however MUSIC robotics is applied, these structures resolve as a cell that compresses a motor axon (h, boxed area) and (i) in higher magnification. (j), (k) this process leaves the myelin sheaths intact.  Figure 2, not recognizable as an emergent structure: if, however, the entire MUSIC robotics data set is used for this cell, this cell exhibits the following mechanism: it displaces a motor axon (Figure 4(i)). Such processes do not exist in the healthy motor system of the pyramidal tract. None of the described cell invasions were found in the postmortem control study with tissue from persons having died suddenly because of heart disease. It can therefore be assumed that cell invasion and consecutive axotomy by the described cell, henceforth called axotomy-competent-cell (ACC), are specific for sporadic ALS.
Progressive motor neuron phenomena of the disease, such as progressive paralysis, breathing insufficiency, and bulbar symptoms are likely to be caused or at least driven by the described cell type. This cell type is therefore referred to as axotomy-competent cell (ACC). The number of ACC per litre blood correlates with the rate of progression of the disease substantiating its proximal pathogenic role.
A MUSIC robotics magnification of an ACC in its active function of axotomizing a motor axon ( Figure 5(b)) shows that the CD3 and the CD8 receptors are polarized in an aberrant distance of 855 nm to each other ( Figure 5(b)), while the requested distance for an antigen recognition-driven transduction of an activating signal into the cell would require a distance less than 1 nm as shown schematically in Figure 5(a). Hence it is unlikely that this cell is able to lyse the axon by an antigen-driven mechanism. This indeed is in strong keeping with the fact that the axoplasm ( Figure 5(b), asterisk) is homogeneous instead of being lysed. Hence, the axotomy is resulting from a displacement process exerted by the ACC, actually an axon-compression. This is in keeping with earlier observations having been made by an initial application of MUSIC robotics: the whole postmitotic system, including the endomysial tube of skeletal muscle fibres is protected from being cytotoxically attacked by invasive T cells, because such cells do not activate early response gene products [13] [16].

Therapeutic Depletion of the ACC
It seems likely that, in the context of postmortem data (Figures 3-5) and recent evidence (3) ACC should be therapeutically destroyed or depleted as early as possible in ALS patients. Since the molecular geometry, respectively the toponome of the ACC in the blood is given [3] (Figure 6(a)), it is conclusive to find these cells in the blood also in a simple blood cell test, under the prerequisite that MUSIC robotics is applied. This was shown to be the case recently [3], and high resolution protein profiling of such ACC (Figure 6 Figure 7 displays the number of ACC in blood over time in a patient treated by ACC depletion. This procedure correlated with the neurological findings briefly described above, showing no pathological spontaneous activity in electromyogram anymore. Another 4 patients with motor deficits in advanced ALS showed the same ACC cells as documented in this case reported here for one patient. Depletion of these ACC was possible also in these more progressed stages of ALS (to be reported in detailed clinical form).
The reason for the therapeutic effect in the one case reported here appears to be that the invasion specificity of the ACC expressing a specific combinatorial molecular structure at the cell surface for the capillary endothelium inside the pyramidal tract (Figure 8) is removed by depletion (Figure 7) or by damaging the functional cell structure of the ACC (Figure 6(c)).

Discussion and Conclusion
The results described here were found by MUSIC robotics microscopy, which was previously used with a combinatorial molecular resolution ranging from 4.31 × 10 86 to 4.4 × 10 481 . The resulting detected high-dimensional geometries in human tissue reveal disease-specific emergence and disease-specific cells in ALS, whose role in this disease can be fully decrypted: the cells whose axotomy competence could be well understood by post-mortem analysis using MUSIC robotics. It led to the conclusion that these cells appear to be causative for the pathogenesis of sporadic ALS: 1) ALS specific cells in the blood are broadly consistent with the CNS-invasive cell type, which compresses motor axons in ALS Figure 7. Illustration of the effect of ACC depletion in a patient. The red line plots the number of ACC (y axis, number of ACC per litre blood) over time (x axis), as detected and controlled by MUSIC robotics. The depletion therapy shows that over long periods of time following depletion therapy the ACC are not detectable any more. On the other hand sudden "eruptions" of ACC numbers can be detected by MUSIC robotics (peak) providing the possibility to enhance the depletion therapy in order to counteract this process.  , and red structure belongs to the cell surface membrane of the blood borne ACC. The blue structure belongs to the internalized CD16 coreceptor of the combinatorial molecular cell surface structure that the ACC has needed for endothelium recognition in a slightly earlier stage. Indeed the shown ACC surface protein combinations (red) and anti combiantions (white spaces ) may represnt an ALS specific cell surface code (red) that recognizes the endothelial address code (yellow). Indeed this is substantiated by the fact that this profile was never found in healthy individuals or patients with other diseases (to be published).
and thereby significantly affects them; 2) if these cells are specifically depleted from the blood circulation, then there is a significant regression of the disease phenomena accompanied by physical well-being (neurologically documented). This association and the fact that they also occur in other ALS patients with the sporadic form of the disease, where the number of these cells per litre of blood correlates with the course rate of the disease, support the conclusion that these cells are causal for the sporadic ALS: Fifty million cells per one litre blood correspond to a threefold slower speed rate of ALS than 150 million cells. Since the patient diagnosed here and treated with cell depletion appears to be clinically healthy again, one can complete the following: the coupling of cycles "MUSIC robotics detection of the cells-then depletion of the cells-control of the measure using MUSIC robotics, etc." seems to be a life-saving measure, if one considers that the disease is always strictly progressive and leads unavoidably to death. The data are compatible with the observation of other investigators, who showed that the molecule TDP-43 in neurons increases and accumulates in ALS [17]. Axotomy is the natural cause of neuronal TDP-43 accumulation (17). However, it cannot be ruled out that sporadic ALS might have several other forms of pathogenesis. However, in the case of sporadic ALS, when ACC is detected, ACC depletion is an indication as a life-saving measure, given the data presented here. The corresponding medical indication should be made as early as possible to prevent irreversible damage of the motor system. The MUSIC robotics technology and corresponding protocols have been described in detail [18]- [24]. The experiences reported here might stimulate a systematic mathematical approach to the molecular geometry of emergence mechanisms in diseases, which cannot be revealed by large scale expression profiling without anatomical resolution, as illustrated in Figure 2, on the right. Translation of the method described here into broad therapeutic application for ALS patients is in preparation.