BIOMATERIALS FOR NEURONAL IMPLANTS AND USE OF SAID BIOMATERIALS IN THE DIAGNOSIS AND THERAPY OF NEURONAL DISEASES

Abstract

The present invention relates to a neural implant comprising a biomaterial having an outer surface with a stochastic nanoroughness (Rq), and the application of said stochastic nanoroughness in the diagnosis and/or treatment of a neurological disorder, such as, for example, Parkinson's disease, Alzheimer's disease, glioblastoma and/or for disrupting and/or preventing glial scars in the context of mammalian mechanosensing ion channels selected from the family of PIEZO-1 and PIEZO-2 ion channels.

Claims

1. Neural implant comprising a biomaterial having an outer surface with a stochastic nanoroughness (Rq) of between 25 and 40 nm, preferably of between 32 nm+/−5 nm, and most preferred of about 32 nm.

2. The neural implant according to claim 1, wherein said biomaterial is selected from platinum, gold, synthetic polymers, for example poly(organo)siloxanes, antimicrobial polymers, polypyrrole (PPy), poly(3,4-ethylene dioxythiophene) (PEDOT), polyterthiophene (PTTh), poly(pyrrole) and its derivatives, Cyclotene®, and parylene C.

3. The neural implant according to claim 1 or 2, wherein said biomaterial further comprises an active substance selected from a pharmaceutically active drug, an antibiotic, a cytotoxic substance, an anti-inflammatory substance, a polypeptide, NGF, BNDF, and collagen.

4. The neural implant according to any of claims 1 to 3, wherein said implant comprises a component selected from a polymer wire, a nanotube, an array of micro-sized posts or pillars, carbon fibers, and composite carbon nanofibers.

5. The neural implant according to any of claims 1 to 4, wherein said implant is permanent or non-permanent and is preferably selected from a measuring and/or stimulating electrode, such as flexible nanoelectrodes, a pacemaker, and a drug delivery device.

6. Method for producing the neural implant according to any of claims 1 to 5, comprising the step of providing a prefabricated neural implant device with a biomaterial having an outer surface with a stochastic nanoroughness (Rq) of between 25 and 40 nm, preferably of between 32 nm+/−5 nm, and most preferred of about 32 nm.

7. The method according to claim 6, wherein said prefabricated neural implant device at least partially consists out of said biomaterial, or wherein said biomaterial is applied as a coating to said prefabricated neural implant device.

8. The method according to claim 6 or 7, wherein said stochastic nanoroughness (Rq) is applied to said biomaterial using a method selected from polishing, machining, surface treatment, coating, cathodic polarization, acid etching, rolling, atmospheric plasma, laser treatment, and casting.

9. Use of the neural implant according to any of claims 1 to 5 for the diagnosis and/or treatment of a neurological disorder, such as, for example, Parkinson's disease, Alzheimer's disease, glioblastoma and/or for disrupting and/or preventing glial scars.

10. A method for identifying a modulator of the activity and/or expression of a mammalian mechanosensing ion channel, preferably selected from the family of PIEZO-1 and PIEZO-2 ion channels, comprising the steps of a) contacting a potential modulator of said mechanosensing ion channel with a cell expressing said mechanosensing ion channel, and b) identifying a modulator of said mechanosensing ion channel by detecting a change in the mechanosensing activity and/or expression of said ion channel in the presence of said modulator, compared to the absence of said modulator.

11. The method according to claim 10, wherein said change in the activity and/or expression is selected from a decrease or an increase of said mechanosensing activity and/or expression of said ion channel.

12. The method according to claim 10 or 11, wherein said mechanosensing ion channel is a cation ion channel, such as, for example, a sodium, calcium or potassium channel, such as, for example, a channel comprising human PIEZO-1 or human PIEZO-2.

13. The method according to any of claims 10 to 12, further comprising testing said modulator as identified for its activity on the interactions of astrocytes with neurons.

14. A screening tool for identifying a modulator of the activity and/or expression of a mammalian mechanosensing ion channel selected from the family of PIEZO-1 and PIEZO-2 ion channels, comprising an isolated cell expressing a recombinant mammalian PIEZO-1 or PIEZO-2 protein, wherein said cell is not a human embryonic stem cell, and wherein said cell is preferably selected from the group of glial cells, astraglia cells, neuronal cells, glioblastoma cells, recombinant mammalian host cells, yeast cells, and bacterial cells.

15. An inhibitor of a mammalian mechanosensing ion channel, preferably of the family of PIEZO-1 and PIEZO-2, for use in the diagnosis and/or treatment of a neurological disorder, such as, for example, Parkinson's disease, Alzheimer's disease, glioblastoma and/or for disrupting and/or preventing glial scars.

Description

[0069] In the Figures:

[0070] FIG. 1 shows a panel of the formation of glial scar tissue in vitro on a smooth glass surface (top panel; Rq=3.5 nm). In comparison, no glial scar tissue is formed on surfaces with a modified topography (bottom panel; Rq=32 nm). Rq is the root mean square roughness.

[0071] FIG. 2 shows (left) the relative amount of glial scar tissue formed in vitro around polymer wires (grey bar) or coated polymer wires with an altered surface topography (black bar). The right figure shows the size of glial scar tissue formed in vitro after re-plating onto plain glass which is a smooth surface (black line) or surfaces with a modified topography, Rq 32 nm (grey line).

[0072] FIG. 3 shows the morphological and functional traits in PC-12 cells on nanorough substrates: (a) Atomic force microscopy (AFM) of silica nanoparticle (SNP) modified substrates with the corresponding surface roughness Rq. (b) Morphology of PC-12 cells on Rq=3.5 nm, Rq=32 nm and Rq=80 nm visualized by staining for F-actin. Impact of nanoroughness on PC-12 polarization as assessed by determining: (c) number of neurites per cell and (d) neurite length. (e) Influence of nanoroughness on acetylcholinesterase (AChE) activity. Calcium sensitive FURA-2 imaging of differentiated PC-12 cells on smooth glass substrates and surfaces with an Rq of 32 nm: (f) change in intracellular calcium levels as assessed by FURA-2 intensity, (g) rate of depolarization as determined by the slope of the depolarization portion of the curve (immediately after addition of KCl). Statistical significance: *p<0.05; **p<0.01; ***p<0.001.

[0073] FIG. 4 shows that the morphology and function of rat hippocampal neurons and astrocytes are influenced by substrate roughnesses: Neuron-astrocyte interaction on (a) smooth glass substrate, (b) on substrate of Rq=32 nm. Astrocytes were visualized using antibody against GFAP (dark gray) and neurons were visualized using antibody against MAP-2 (light gray). Quantification of neuron-astrocyte association in: (c) short-term cultures (5 days), and (d) long-term cultures (6 weeks). Calcium sensitive FURA-2 imaging in hippocampal neurons on smooth glass substrates and surfaces with Rq of 32 nm: (e) change in intracellular calcium level as assessed by FURA-2 intensity, (f) rate of depolarization as determined by the slope of the depolarization portion of the curve (immediately after addition of KCl). Statistical significance: ***p<0.001.

[0074] FIG. 5 shows that piezo-1 is necessary for sensing nanotopography: (a) Representative scanning electron micrograph of PC-12 cells grown on nanorough surface (shown Rq=40 nm). PC-12 stained using anti-FAM38A, an antibody for Piezo-1 mechanosensitive ion channel on Rq=3.5 nm (b) and Rq=32 nm (c). On smooth surfaces, FAM38A staining is pronounced at neurite branch points (denoted by circles) (b), while on nanorough surfaces FAM38A staining is uniform along all neurite processes (c). Rat dorsal root ganglia morphology (d, e) and function (f, g) on glass and Rq of 32 nm. Inhibition of FAM38A with GsMTx4 (5 μM) results in decoupling of hippocampal neurons from astrocytes on smooth glass substrates (h, i). The FURA-2 intensity profile (j), and rate of calcium influx (k) in hippocampal neurons upon depolarization with KCl on smooth glass substrate and Rq=32 nm is identical upon inhibition of FAM38A. Statistical significance: ***p<0.001.

[0075] FIG. 6 shows that nanoroughness alters physical attributes of astrocytes: Dependency of astrocyte form factor on substrate nanoroughness: (a) Decrease in form factor on 32 nm Rq surfaces is consistent with a more motile phenotype (inset). Morphological changes to astrocyte cell surface on nanorough surfaces: AFM images of astrocytes grown on glass (b) and on Rq=32 nm (c) and the corresponding transverse line scans, and changes to topography of astrocyte surface on 32 nm surfaces (d). Shaded areas show representative areas for Rq calculations. Amyloid-beta plaques are associated with topographical changes to brain tissue: Paraffin embedded human brain slices stained with Bielschowsky's silver stain (e): Left image; healthy human (AD−), Right image; patient diagnosed with Alzheimer's (AD+): revealing amyloid β plaques indicated by yellow arrowheads. Bottom panel; tapping mode AFM scans (10 μm×10 μm) of one of the representative areas (top), higher magnification (2 μm×2 μm) scan of the same area (bottom), and transverse views of the corresponding 2 μm×2 μm image above. (f) Histograms of Rq values of healthy brain tissue, and amyloid-β plaques in Alzheimer's patients showing a general shift of tissue roughness to higher Rq's and an increased heterogeneity in roughness in AD+ brain slices. Rq values were calculated using a 700 nm×700 nm scan area.

[0076] FIG. 7 shows the results of the siRNA knockdown of FAM38 in Astrocyte/Hippocampal neuron co-cultures (see Example 3).

[0077] FIG. 8 shows glial scar formation in vitro using meningeal fibroblasts+cortical astrocytes+TFG-beta-1 (see Example 4 and also FIG. 1).

[0078] FIG. 9 shows glial scar dissociation upon replacing of existing scar clumps onto Rq=32 nm surfaces. (A) Optical micrographs, (B) Change in sphere size as a function of time (see also FIG. 2).

[0079] FIG. 10 shows AFM of dip-coated electrodes with an Rq of 32 nm before (left) and after (right) “implantation” into agarose gels for 4 weeks, and rheology of different agarose gels.

[0080] FIG. 11 shows glial scar formation on non-coated (top) vs. coated (bottom) polymer wires (A), and quantification of scar built-up (B) (see also FIG. 2).

[0081] FIG. 12 shows an optical micrograph of 15-pol electrode (A), AFM of 15-pol electrode dip-coated with silica nanoparticles to create a surface roughness of Rq=32 nm (B), and impedance measurements before (C), and after nanoparticle coating (D).

[0082] FIG. 13 shows the formation of sprouts from GMB tumor spheroids, which is an indication of invasiveness migration in the presence or absence of the spider venom toxin GsMTx4 which inhibits Piezo-1.

[0083] FIG. 14 shows that using glioblastoma multiforme (GBM) cell lines and primary GBM cells from human tumor explants, their migration in a trans well migration assay can be completely stopped by knocking down piezo-1. GBM=GBM-1; KF=GBM-2.

EXAMPLES

Example 1

[0084] The properties of the inventive biomaterial surface were tested and verified in a clinically relevant experimental model. In the in vitro model of glial scarring, polymer wires with the surface coating were able to inhibit glial scar formation. Moreover, already (in vitro) formed glial scar tissue decomposed when exposed to a specific regimen of nanotopography as it is used for the coating. Polymer wires with surface coating were implanted into Agarose gels with an almost similar consistency as brain tissue, and the coating was shown to be stable after this implantation (see FIGS. 1 and 2).

[0085] The surfaces of the materials can be characterized for morphology and roughness using scanning electron microscopy (SEM).

Example 2

[0086] Nanotopography Modulates PC-12 Cell Polarity and Enhances Function

[0087] Since macromolecules are in a state of high entropy, and entropy is a statistical measure of randomness, the roughness presented by macromolecules is expected to be stochastic (random). The inventors simulated random ECM nanoroughness using an assembly of monodispersed silica colloids of increasing size (Lipski A M, Pino C J, Haselton F R, Chen I-W, Shastri V P (2008) The effect of silica nanoparticle-modified surfaces on cell morphology, cytoskeletal organization and function. Biomaterials 29:3836-46, Lipski a. M et al. (2007) Nanoscale Engineering of Biomaterial Surfaces. Adv Mater 19:553-557) (FIG. 3a). The roughness in this system scales logarithmically with nanoparticles radius and can recapitulate topography from the level of receptor clusters to ECM features (Shastri V P (2009) In vivo engineering of tissues: Biological considerations, challenges, strategies, and future directions. Adv Mater 21:3246-54), and further allows the production of surfaces with stochastic nanoroughness. In contrast, surfaces consisting of periodic groves and ridges that have been extensively studied present deterministic roughness.

[0088] As a first step, the inventors investigated the ability of PC-12 cells (Greene L, Tischler S (1976) Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc Natl Acad Sci USA 73:2424-8), a well-established model system for studying neuronal differentiation, to perceive stochastic nanoroughness and analyzed changes to their morphology and function.

[0089] PC12 cells are indeed able to perceive the underlying nanoroughness (in an NGF- and collagen-dependent manner) and showed an increased differentiation and associated functional traits on a specific Rq of about 32 nm as evident from a highly polarized cell morphology (FIG. 3b, middle panel) and associated changes such as fewer and longer neurite outgrowths (FIGS. 3c and 3d) compared to glass, which is considered a smooth substrate (Rq approx. 3.5 nm, FIG. 3b, left panel). Beyond this optimal substrate roughness, cells were more prone to clumping (FIG. 3b, right panel). If the slides or culture substrates is not coated with collagen, then the PC-12 cells cannot sense the roughness, and also they need NGF in order to express neurites which are the sensing elements for sensing the nanotopography. For growing neuronal cells, in general one has to coat the substrate with either collagen polylysine or other polycation, such as poly-L-ornithine. Nevertheless, this coating does not affect the roughness and/or Rq values.

[0090] One measure of the functional state of a neuron is the activity of acetylcholinesterase (AChE) as this is necessary for synaptic communication. Interestingly, AChE levels also peaked in PC-12 cells on 32 nm Rq surfaces (FIG. 3e), which also coincided with an accelerated and elevated calcium response to depolarization (FIGS. 3f and 3g).

[0091] Nanotopography Mediates Hippocampal Neuron-Astrocyte Interaction

[0092] The inventors then posed the following question: can neuronal cells in general perceive nanoroughness and, if so, does it have a role in defining their interaction and function? Hippocampal neurons are responsible for memory formation. Loss of their function and death has been linked to neuropathologies, such as Parkinson's and Alzheimer's disease.

[0093] The inventors therefore evaluated the response of mixed primary cultures of rat hippocampal neurons and astrocytes to the different roughness regimes. Surprisingly, primary hippocampal neurons also responded to roughness in a manner similar to dopaminergic PC-12, and exhibited prominent, axon like polarized structures on exactly the same Rq of 32 nm.

[0094] An also remarkable finding was that nanoroughness appeared to modulate the relationship, and dependency of neurons on astrocytes. It is well established that neurons require astrocytes for survival (Cui W, Allen N D, Skynner M, Gusterson B, Clark a J (2001) Inducible ablation of astrocytes shows that these cells are required for neuronal survival in the adult brain. Glia 34:272-82), and indeed, on surfaces with an Rq above and below 32 nm, neurons were predominantly found associated with astrocytes (FIGS. 4a and 4c). However, on Rq of approx. 32 nm, neurons were dissociated from astrocytes (FIGS. 4b and 4c) and continued to survive independently even up to 6 weeks (FIG. 4d). After 5 days, the percent neurons that were associated with astrocytes on the 32 nm Rq surface was around 15%, which was 1.5-2 fold lower than those on other Rq's, which ranged from 20-40%, and 6 fold lower than that on the smooth glass substrate (FIG. 4c). That is, in comparison to the other Rq's, over twice as many neurons on 32 nm Rq surface were surviving independently of astrocytes. At 6 weeks however, the percent of neurons that were surviving independently of astrocytes on the 32 nm Rq was over 6 fold greater in comparison to the other Rq's. While over 90% of neurons were associated with astrocytes on other roughnesses, only 15% of neurons were associated with astrocytes on 32 nm Rq (FIG. 4d). Remarkably, hippocampal neurons on 32 nm Rq surfaces in spite of being dissociated from astrocytes showed an order of magnitude faster and stronger increase in intracellular calcium levels following membrane depolarization in comparison to those on smooth surfaces (FIGS. 4e and 4f). Thus, there seemed to be a favorable Rq of around 32 nm at which both PC-12 and hippocampal neurons appeared to be more functional.

[0095] Mechanosensing Ion Channel—Piezo-1 is Responsible for the Sensing of Nanoscale Physical Cues by Neurons

[0096] Past studies showed that stochastic nanoroughness altered the organization of focal adhesion complexes in highly migratory preosteoblasts and endothelial cells (A M Lipski, C Pino, F R Haselton, I-W. Chen, and V P Shastri; “The effect of silica nanoparticle-modified surfaces on cell morphology, cytoskeletal organization and function”, Biomaterials, (28), 3836 (2008)). Since neurons have limited migratory capacity (Fricker R a et al. (1999) Site-specific migration and neuronal differentiation of human neural progenitor cells after transplantation in the adult rat brain. J Neurosci 19:5990-6005), a critical open question was how neurons perceive nanoroughness. Scanning electron micrographs revealed that the neurites indeed make intimate contact with the underlying topography (FIG. 5a). Such intimate contact between the neurites and the surface ought to manifest itself as changes in membrane tension. Since the conformation and distribution of mechanosensitive ion channels is altered in response to changes in membrane tension and curvature (Nilius B (2010) Pressing and squeezing with Piezos. EMBO Rep 11:902-3), the inventors investigated the expression pattern of FAM38A, an integrin-activated transmembrane protein, which is part of the mechanosensitive ion channel Piezo-1 (Coste B et al. (2010) Piezo1 and Piezo2 are essential components of distinct mechanically activated cation channels. Science 330:55-60). Piezo-1 is expressed by CNS neurons and not by sensory neurons like dorsal root ganglia (DRG) (Roudaut Y et al. (2012) Touch sense: functional organization and molecular determinants of mechanosensitive receptors. Channels 6:234-45).

[0097] It was observed that, while FAM38A expression in PC-12 cells on glass was predominantly localized at neurite branch-points which would be a region of high cytoskeletal tension (FIG. 5b), in contrast, a more uniform distribution of FAM38A could be seen on the optimal Rq of 32 nm, suggesting a dramatic change to the mechanical environment of the neurite as they perceive the nanoroughness (FIG. 5c). Since FAM38A expression level was not altered, and PC-12 during differentiation did not show any changes in attachment force or motility in response to nanoroughness, the observed changes to Piezo-1 expression pattern can be linked to the underlying nanotopography.

[0098] The role of Piezo-1 in sensing topography is further bolstered by the findings that DRGs, which lack this mechanosensitive channel, but possess Piezo-2 instead, do not show any morphological changes on nanoroughness substrates (FIGS. 5d and 5e). This is further confirmed by imaging the calcium flux, which showed similar depolarization patterns and rate of calcium influx in DRGs grown on Rq of 3.5 and DRGs grown on Rq of 32 nm (FIGS. 5f and 5g).

[0099] Neuron-Astrocyte Interactions Involve Topographical Cues Provided by Astrocytes and Piezo-1

[0100] As indicated above, primary hippocampal neurons require the interaction with astrocytes for their survival (Cui W, Allen N D, Skynner M, Gusterson B, Clark a J (2001) Inducible ablation of astrocytes shows that these cells are required for neuronal survival in the adult brain. Glia 34:272-82). This raised the question as to why do the neurons favor the surface over association with the astrocytes. AFM analysis of the surface of astrocytes associated with neurons led the inventors to the remarkable finding that the roughness of the astrocyte surface was around an Rq of 26-28 nm (FIG. 6d), and this coincides rather well with the roughness regime on which neurons exhibit decoupling from astrocytes.

[0101] A role of mechanotransduction in maintaining neuron-astrocyte interactions is further supported by the inventors' findings that upon inhibition of Piezo-1 with the toxin GsMTx4 (Delmas P, Hao J, Rodat-Despoix L (2011) Molecular mechanisms of mechanotransduction in mammalian sensory neurons. Nat Rev Neurosci 12:139-53, Bae C, Sachs F, Gottlieb P (2011) The mechanosensitive ion channel Piezo1 is inhibited by the peptide GsMTx4. Biochemistry 50:6295-6300), neurons decouple from astrocytes even on smooth glass substrates (FIGS. 5h and 5i) where their normally show strong association (FIGS. 4a, 4c, and 4d). Furthermore, the increased sensitivity to depolarization that was observed in hippocampal neurons on Rq of 32 nm is lost upon inhibition of Piezo-1 (FIGS. 5j and 5k). This provides direct evidence for the role of nanotopography is influencing hippocampal neuron-astrocyte interaction and function through mechanotransduction, and a clear role for stretch-activate ion channels in these processes.

[0102] Regions of Amyloid Plaque Build-Up in Alzheimer's Present Increased Tissue Nanoroughness

[0103] The inventors' observation that topography of astrocytes, a support cell for neurons, can dictate the function of the phenotypically unrelated neurons, points to a larger paradigm wherein physical and mechanical information provided by astrocytes and ECM macromolecules play a role not only in neuronal development but also in neuropathologies. There is ample evidence that the loss of memory associated with Alzheimer's disease (AD) is due to the death of hippocampal neurons. PGs like chondroitin sulfate PGS (CSPGs) have been implicated both in neural differentiation and neuropathologies such as Alzheimer's (Galtrey C M, Fawcett J W (2007) The role of chondroitin sulfate proteoglycans in regeneration and plasticity in the central nervous system. Brain Res Rev 54:1-18). CSPGs have been found to co-localize with amyloid-β plaques, and in vitro studies have shown that CSPGs can promote amyloid-β fibril assembly a key step in plaque formation. Interestingly, amyloid-β stimulates CSPG production in astrocytes, which has negative effects on neuronal health and synapse formation.

[0104] Thus, the loss of hippocampal neuron function in Alzheimer's seems to be triggered by changes to the topography that the neurons experience. In order to investigate this premise further, the inventors analyzed the topographical characteristics of amyloid-β plaques in the hippocampus of human brain slices using AFM (FIG. 6e). The inventors made the compelling observation that while the Rq of healthy brain tissue (AD−) showed a Gaussian distribution with a median centered around 34 nm, the tissue of individuals diagnosed with Alzheimer's (AD+) showed a bimodal distribution of tissue roughness with a pronounced shift in the median towards higher Rq values of 60 nm accompanied by a more heterogeneous Rq pattern (FIG. 6f). The emergence of Rq values greater than 80 nm, which is the range of Rq where the inventors observe increased neuronal cell death, is in accordance with published reports that neurons, when exposed to amyloid-β undergo apoptosis (Fraser P E, Lévesque L, McLachlan D R (1994) Alzheimer A beta amyloid forms an inhibitory neuronal substrate. J Neurochem 62:1227-30; Ivins K J, Thornton P L, Rohn T T, Cotman C W (1999) Neuronal apoptosis induced by beta-amyloid is mediated by caspase-8. Neurobiol Dis 6:440-9). The current observation that astrocyte shape is affected by nanoroughness provides evidence that cell signaling in the neuronal environment may be additionally mediated by ECM-based cues.

[0105] The effects of stochastic nanoroughness on neuronal health seem to manifest itself in two possible scenarios: (1) The changes to tissue roughness affects glial cell behavior which then instigates changes to neuron signaling environment, and/or (2) the changes to generally stationary cells that provide a supportive network for neuronal cells and synapses, migratory and proliferating astrocytes have been observed in glial scarring, an environment with diminish neuronal function (Buffo A, Rolando C, Ceruti S (2010) Astrocytes in the damaged brain: molecular and cellular insights into their reactive response and healing potential. Biochem Pharmacol 79:77-89, Wanner I B et al. (2013) Glial scar borders are formed by newly proliferated, elongated astrocytes that interact to corral inflammatory and fibrotic cells via STAT3-dependent mechanisms after spinal cord injury. J Neurosci 33:12870-86). In the present invention, an altered cellular environment in the form of nanotopography was shown to affect astrocyte biophysical attributes (shape, roughness) so as to alter its interaction with neurons.

[0106] Strong evidence for the second scenario is derived from a recent study by Satoh et al. (Satoh K et al. (2006) A novel membrane protein, encoded by the gene covering KIAA0233, is transcriptionally induced in senile plaque-associated astrocytes. Brain Res 1108:19-27), showed that hMib (a human ortholog of rodent Piezo-1) is transcriptionally induced in activated astrocytes associated with senile amyloid-β plaques in AD+ human brains. Interestingly, neurons that express hMib show damaged morphology while healthy looking neurons do not express hMib. The ability to sense the changes to astrocyte topography induced by tissue roughness seems to have triggered undesirable changes in hMib+ neurons. Conversely, the inability to sense the mechanical cues provided by the astrocytes seems to play a role in the loss of function in the hMib− neurons. Since healthy neurons are hMib+, loss of this marker seems to additionally play a role in the functional deficiency associated with Alzheimer's.

Example 3

[0107] Piezo-1 Knockdown Using siRNA

[0108] Scanning electron microscopy: For imaging cells grown on nanoparticle modified surfaces, cells were fixed in 4% PFA for 30 minutes, washed with PBS and dehydrated in an increasing ethanol gradient followed by a drying phase in vacuum. Imaging was accomplished with a Quanta 250 FEG (FEI Inc.) equipped with the FEI×T software.

[0109] Atomic force microscopy: Scans were done in tapping mode with a Dimension V atomic force microscope (Bruker Ltd.) equipped with the Nanoscope software (V.7.3, Bruker Ltd.). Nanoparticle modified surfaces and Alzheimer's disease (AD) samples were measured with a phosphorus-doped silica cantilever in air (k=3 N/m, f.sub.0=74-90 kHz) at a scan rate of 0.9 Hz with 256 lines per image. Per batch of coated SNPs, three different substrates were analyzed and the root mean square roughness R.sub.q calculated for three independent regions of each substrate. Amyloid-β plaque roughness was determined for a total of 100 areas (from two individual patients per condition) positively stained in the silver staining (as described see below).

[0110] Cells were fixed in 4% PFA for 30 minutes, washed with PBS and imaged in water with the help of a fluid cell. Scan were done with a silicone tip on nitride lever (k=0.32 N/m, f.sub.0=40-75 kHz) at 512 lines per image with a scan rate of 0.312 Hz. For astrocyte cell surface roughness measurements, 15 cells from 3 different animals were measured. For R.sub.q calculations of the cell surface, a total of 150 areas were analyzed.

[0111] Immunocytochemistry: Cells were fixed for 30 minutes with 4% paraformaldehyde before antibody specific staining. Postmitotic neurons were visualized with an anti-MAP-2 antibody (Abeam), early stage neurons with an anti-neuron specific class III β-tubulin antibody (TuJ-1, Abcam) and astrocytes with an anti-GFAP antibody (Dako GmbH). The stretch activated ion channel Piezo-1 was identified with an anti-FAM38A antibody (Abeam Inc.). Visualization of the actin cytoskeleton was done with phallotoxin conjugated to Alexa Fluor488 (Invitrogen Life Technologies GmbH, Germany).

[0112] Cell density/composition analysis: In order to analyze the cell density and composition of mixed neuron/astrocyte cultures on the different substrates at the end of the experiments, MAP-2.sup.+, GFAP.sup.+ and Tuj-1.sup.+ cells were counted in 4 separately isolated cultures in 10 random image sections and on two different substrates of each isolated culture.

[0113] Morphological analysis: Morphological changes in cells grown on SNP modified substrates were analyzed in biological and technical triplicates. Per condition and substrate 20 random pictures were used for evaluation of morphological changes. For PC12 cells, only those not contacting other cells, and only processes extending from the cell soma with a length bigger than the cell's diameter were used for analysis. The astrocyte form factor was analyzed with the help of ImageJ (Fiji V.1.47p, NIH, USA) and calculated according to the following formula:

[00001]$\phi =\frac{4.Math.\pi .Math..Math.A}{p^2}$

Where, A is the cell's diameter and p the cell's perimeter. Values for the form factor can be between zero and one, zero almost being a line and one being a perfect circle.

[0114] Small interfering RNA knockdown experiment: For all transfection experiments, primary hippocampal neuron/astrocyte cultures were seeded as 7.5*10.sup.4 cells/cm.sup.2 and cultured in serum containing media. Rat FAM38A small interfering RNA (On-Target Plus, Dharmacon), control small interfering RNA (AllStars negative siRNA, Qiagen) and/or the transfection reagent DharmaFect 3 (Dharmacon) was added to the culture in varying concentrations after 24 h in serum free Neurobasal media supplemented with B27 and glutamine and according to the manufacturer's instructions. The siRNA sequences targeting FAM38A were the following:

TABLE-US-00001 (SEQ ID No: 1) 5′-GCACAAAGGCCUCCGACUU-3′, (SEQ ID No: 2) 5′-GGGUUGAAGAUUCGGGAGA-3′, (SEQ ID No: 3) 5′-CGGAAGAAUGGCAGCGCAU-3′,  and (SEQ ID No: 4) 5′-CAGAUGAACAGUUGGGCGA-3′.

[0115] Knockdown efficiency was assessed by quantitative real-time PCR.

[0116] Calcium sensitive imaging: Intracellular calcium levels were measured with the cell permeable probe FURA-2-acetoxymethyl ester (Invitrogen Life Technologies GmbH). Cells were exposed to 5 μM FURA-2-AMin DMSO (final concentration of 0.2%) for 45 minutes in a humidified incubator at 37° C. Increase in intracellular calcium levels following depolarization with 50 mM KCl was analyzed by the change in the absorption and emission spectra of FURA-2 upon Ca.sup.2+ binding using a custom-built perfusion chamber mounted to a Zeiss Observer Z1 and the ZEN blue software (Zeiss AG, Germany). The rate of intracellular Ca.sup.2+ increase was calculated as the slope of the linear portion of the increase in FURA-2 intensity.

[0117] Acetylcholinesterase activity measurement: PC12 cells were detached from the substrate by trypsin treatment and washed twice with PBS. Cells were resuspended in 0.1M Na-phosphate buffer (pH 8.0) containing 1% Triton X-100 and sonicated for 20 seconds. For enzyme activity measurement, 5 μl cell homogenate was mixed with 190 μl of a 10 mM dithiobisnitrobenzoic acid solution (DTNB, Sigma) and 5 μl were transferred into a 96-well plate. After addition of 5 μl acetylthiocholine iodide (final concentration 0.5 M, Sigma) the change of absorbance at 412 nm was followed for 10 minutes.

[0118] Immunohistochemistry of AD slices: Slices of patients diagnosed with Alzheimer's disease and patients of the same age diagnosed negative for Alzheimer's disease were provided by the University Hospital Freiburg and were in obtained in accordance with institutional ethical guidelines. Paraffin embedded samples were stained according to the Bielschowsky's silver staining. Slices were deparaffinized and incubated in 10% silver nitrate solution for 15 minutes. After washing, samples were incubated for 30 minutes at 30° C. in an ammonium silver solution, treated with a developing solution followed by one minute incubation in 1% ammonia hydroxide to stop the silver reaction. After washing, slices were imaged immediately with a Zeiss Observer A1 (ZEISS AG, Germany) and with the AFM as described above.

Example 4

[0119] a) Inhibition of the Formation of Glial Scars

[0120] Glial scars are formed in vitro in a model system using meningeal fibroblasts and cortical astrocytes in the presence of TGF-beta. When these cells were plated on a surface with a nanoroughness of 32 nm Eq, they did not organize into glial scars (see also FIG. 1).

[0121] For the formation of glial scars in vitro, two primary cell types (meningeal fibroblasts and cortical astrocytes freshly isolated from rats) were seeded together in a cell culture dish on opposite sides of the dish. Once the cells grew together, TGF-β was added to the culture, and scar tissue formed within 24 hours. Afterwards these were stained with DAPI (visualizing the cell nucleus) and antibodies against GFAP (visualizing the astrocytes) and fibronectin (visualizing the fibroblasts). The SEM (scanning electron microscopy) shows the complete scar tissue for a better understanding of its morphology (FIG. 8).

[0122] b) Dissolution of Glial Scars

[0123] Pre-formed glial scars were then taken and placed on the 32 rq nanorough surface. After 24 hours, a dissociation was observed (see FIGS. 9 and 2).

[0124] For the upper part of FIG. 9, cells, glial scars were generated as described above (on glass; Rq of glass=3.5 nm) and transferred onto another glass substrate, and observed over time. The lower scar tissue was also generated as described above, and transferred onto a surface with a nanoroughness of 32 nm. It can be seen that the size of the scar tissue decreases when it was transferred onto 32 nm substrates. After ˜80 hours incubation, most of the scar tissues are ‘dissolved’, and it seems that cells are actively growing out of the scar tissue. This observation is quantified in the graph below (FIG. 9) indicating that scar tissue that was re-seeded onto glass also very slightly reduced in size. However, scar tissue re-seeded onto Rq=32 nm dissolved in the majority of cases.

[0125] Both these findings are significant with respect coatings for neural implants and treatment of spinal injuries as they show that stochastic nanoroughness in a certain regime can both mitigate formation of glial scar, but also absolve the formed glial scar.

Example 5

[0126] Coating of Electrodes and Wires for Implantation (See FIGS. 10 to 12)

[0127] Electrodes (15-pol microelectrodes) were dip-coated with SNPs thus creating an electrode surface roughness of 32 nm. This was confirmed with AFM (a). These electrodes were then ‘implanted’ into agarose gels for 4 weeks. These agarose gels had the same shear modulus as normal brain tissue has (c). In order to verify the coating integrity of the electrodes AFM was performed again (b). This experiment should show that coating microelectrodes with SNPs can persist implantation into brain (as is the supposed usage) while keeping the coating intact (FIG. 10).

[0128] FIG. 12 shows (A) a picture of the 15-pol microelectrode as mentioned above. (B) SNP coating of the electrode and AFM scanning micrograph of the same as described above. (C) Impedance of the electrodes before (black graphs) and after (red graphs) coating with SNPs. (D) Theta-measurement of the electrodes before (black graphs) and after (red graphs) coating with SNPs. The graphs show that coating the electrodes with SNPs does not alter the impedance or the theta of the electrodes, thus not changing their behaviour regarding the intended use as brain stimulators.

[0129] As shown in FIG. 11, (a) glial scars were created as described above. A polymer wire was introduced into the cell culture dish (the white dotted line represents the position of the polymer wire before removing it for better visualization of the cells). This wire was either coated with SNPs (thus having a surface roughness of Rq=32 nm) or not coated. Over a variety of experiments, significantly more scar tissue was formed on non-coated polymer wires compared to coated polymer wires with a surface roughness of 32 nm (b).

Example 6

[0130] U-87 Spheroid Matrigel Invasion Assay

[0131] The formation of sprouts from GMB tumor spheroids which is an indication of invasiveness (Invasion assay) was significantly reduced in presence of the spider venom toxin GsMTx4 which inhibits many cation channels, including Piezo-1 (FIGS. 13, and 5).

[0132] Spheroids (which resemble a GBM tumor in vitro) are formed via the hanging drop procedure. For this, cells were seeded into a carboxymethylcellulose solution (with very high viscosity) and pipetted on the inner side of the lid of a cell culture plate. After reversing the lid and putting it back onto the cell culture plate, the cells are hanging in a drop of carboxymethylcellulose. Because of the high viscosity of the media and no possibility to attach, the cells form spheroids. These spheroids were washed and re-seeded into a 3D matrix of collagen I and carboxymethylcellulose. During incubation cells start migrating out of the spheroid and invading the 3D matrix. The higher the invasive potential of the cells, the more they will invade the 3D matrix. It is obvious that cell have less invasive potential when GsMTx4 (a spider venom that blocks mechanosensitive channels) is added to the culture media.

Example 7

[0133] Knock-Down of Piezo-1

[0134] In a second assay, regarding Piezo-1, using glioblastoma multiforme (GBM) cell lines and primary GBM cells (KF) from human tumor explants of various aggressiveness, it was shown that their migration (through matrigel/collagen in a trans well migration assay) can be completely stopped by knocking down (silencing) piezo-1 (see FIGS. 7, and 14).

[0135] The experimental set-up was a classical transwell migration assay. A 24-well plate insert with a membrane (8 um pore size) was coated with Matrigel, forming a 3D matrix. After Matrigel was jellified, cells (U-87, GBM (GBM-1), KF (GBM-2)) were seeded in the top of the Matrigel (the media in which the cells are seeded was either supplemented with siRNA against FAM38A or without any additional agent). Below the membrane was medium, either supplemented with an attractant (in this case the chemoattractant was FBS) or no attractant. Over a time of 24 hours, the cells were incubated and the ones that migrated through the Matrigel and attached to the membrane were stained with DAPI and quantified. The bigger the amount of migrated cells, the bigger their migratory potential). The graph shows that with chemoattractant and without siRNA the cells strongly migrate through the Matrigel. Without chemoattractant, less cells migrate through, and this movement was only due to random motion of the cells. The use of FAM38A-siRNA clearly reduced the migratory potential of all three cells lines used. Since the extraordinary high capacity of glioblastoma cells to migrate and invade surrounding tissue is the hallmark of GBM, reducing this capacity indicate an interesting pharmaceutic target, i.e. provides evidence for a use of Piezo-1 as a target for treating brain tumors and especially GBM.