KR20200086277A - Dementia treatment using visual stimulation to synchronize gamma vibrations in the brain - Google Patents

Abstract

Apparatus, systems and methods for treating dementia or Alzheimer's disease in a subject in need thereof. In one embodiment, chronic vision with a frequency of about 30 Hz to about 50 Hz, more specifically about 40 Hz to enhance gamma vibration in multiple brain regions of a subject, including the prefrontal cortex (PFC) and hippocampus Irritation is transmitted non-invasively to the subject. Tuned gamma vibration induces various neuroprotective effects (e.g., improved amyloid plaque and tau hyperphosphorylation) and modulates neuronal activity across multiple brain regions to reduce neurodegeneration (e.g. neural networks at low gamma frequencies) Facilitates functional coupling of). Neural activity mediated by chronic visual stimulation reduces immune responses in microglia and improves abnormally modified genes and proteins associated with membrane transport, intracellular transport, synaptic function, neuroinflammatory and DNA damage responses. Behavior modification, including improved learning and memory, is observed.

Description

Dementia treatment using visual stimulation to synchronize gamma vibrations in the brain

Cross reference to related applications

This application claims the benefit of priority for each of the following U.S. patent provisional applications: No. 62/570,250 (filed on October 10, 2017) "NEUROPROTECTIVE EFFECTS OF COMBINED SENSORY STIMULATION" (Patent Attorney Document Number MITX-9699/00US ); And 62/570,929 (filed October 11, 2017) "GAMMA ENTRAINMENT BINDS HIGHER ORDER BRAIN REGIONS AND OFFERS NEUROPROTECTION" (Patent Attorney Document Number MITX-0070/00US). Each of these provisional applications is incorporated herein by reference in its entirety. In addition, this application claims the priority benefit of PCT Application No. PCT/US18/51785 filed "Systems and Methods for Preventing, Mitigating, and/or Treating Dementia" filed on September 19, 2018, the entirety of which is incorporated by reference. As incorporated herein.

Government support statements

The present invention was made with US government support under grant number RF1 AG054321 awarded by the National Institutes of Health. The United States government has certain rights in this invention.

Dementia, including Alzheimer's disease (AD), is a fatal disease of the brain characterized by deterioration of the brain and cognitive function (Canter et al., 2016; Palop and Mucke, 2016). Several factors contribute to the pathogenesis of AD, including amyloid-β deposition, hyper-phosphorylated tau accumulation, microglia- and astrocytic-mediated inflammation, and loss of neurons and synapses (Ballatore et al., 2007; Huang and Mucke , 2012; Jacobsen et al., 2006; Meyer-Luehmann et al., 2008; Oakley et al., 2006; Ulland et al., 2017; Yoshiyama et al., 2007).

More recent studies are further investigating the physiological aspects of AD pathology such as nervous over-excitation, combined neuronal dysfunction, shifted suppression/excitation balance, epilepsy discharge, and altered network vibration (Canter et al., 2016; Holth et al. 2017; Hsia et al., 1999; Palop and Mucke, 2010; Palop and Mucke, 2016; Verret et al., 2012). The results of these studies are consistent with the network anomalies observed in human AD (Guillon et al., 2017; Koenig et al., 2005; Ribary et al., 1991; Stam et al., 2002). A recent study of the AD mouse model emphasized that this change occurred at the presymptomatic stage (Gillespie et al., 2016; Iaccarino et al., 2016). Changes in neuronal activity have previously been shown to affect AD pathologies such as amyloid-β and tau accumulation in several mouse models (Bero et al., 2011; Wu et al., 2016; Yamada et al., 2014). Given these observations, several approaches have been used to investigate whether manipulating neuronal vibration can be effective in improving AD pathology (Iaccarino et al., 2016; Martinez-Losa et al., 2018; Verret et al., 2012). ).

In particular, it has been found that vibration in the gamma frequency band (about 30-90 Hz) is reduced in several AD mouse models, including hAPP-J20, ApoE4, and 5XFAD, as well as in human AD patients in particular (Gillespie et al., 2016; Guillon et al., 2017; Iaccarino et al., 2016; Koenig et al., 2005; Ribary et al., 1991; Stam et al., 2002; Verret et al., 2012). In view of this, several recent studies have targeted gamma vibration, and the results suggest that this may represent a promising strategy to alleviate AD pathology.

In one previous approach, increased gamma vibration is achieved through expression of a voltage-gated sodium channel subunit Nav1.1 in a Fabalbumin-positive (PV+) associated neuron, or by brain implantation of a Nav1.1 overexpressing associated neuron progenitor cell. It alleviated gamma deficiency in hAPP-J20 mice and decreased both epileptic activity and cognitive decline (Martinez-Losa et al., 2018; Verret et al., 2012).

In a second approach, the optogenetic activity of PV+ associated neurons (Cardin et al., 2009; Sohal et al., 2009) at 40 Hz, which has been shown to induce strong gamma frequency oscillations, reduces amyloid load in 5XFAD mice and is associated with microglia. It has been found to improve morphological transformation (Iaccarino et al., 2016). This non-invasive approach using 40 Hz visual stimulation was similarly effective in reducing amyloid load and altering microglia in the visual cortex of 5XFAD mice (Iaccarino et al., 2016). However, in this previous study, amyloid levels in the visual cortex returned to baseline 24 hours after acute visual stimulation for 1 hour. However, previous studies have shown that prolonging 1 hour of visual stimulation to 1 hour of visual stimulation per day for 7 days is a soluble and insoluble form of amyloid levels (Aβ1-40 and Aβ1-42) in the visual cortex of 6 month old 5XFAD mice. ), as well as reducing plaque pathology (Iaccarino et al., 2016). In addition, visual stimulation affects several cell types (including neurons and microglia) that reduce Aβ production and enhance clearance, respectively, in 5XAFD mice.

As disclosed in U.S. Patent Application No. 15/360,637, filed on November 23, 2016 under the name "SYSTEMS AND METHODS FOR PREVENTING, MITIGATING, AND/OR TREATING DEMENTIA", the entirety of which is incorporated herein by reference. , Inducing synchronized gamma vibrations in the brain through visual (as well as auditory and/or tactile) stimulation reduces amyloid load and morphological changes in some brain regions. However, the present inventors still need a system and method for treating diseases across circuits that affect a number of brain centers that are particularly responsible for learning and memory and other higher-order brain functions as systems and methods for treating dementia and Alzheimer's disease. Recognized and understood that

In the present disclosure, the present invention for synchronizing gamma vibration in the brain of a subject through chronic non-invasive visual stimulation, referred to herein as "Gamma ENtrainment Using Sensory visual stimuli" (GENUS) The methods and tools not only extend low gamma interference to a number of different brain regions beyond the visual cortex (eg, hippocampus, somatosensory and prefrontal cortex), but also enhance low gamma interference across these multiple brain regions at the same time. Has been proven. In addition, chronic GENUS reduced neurodegeneration in 5XFAD, P301S and CK-p25 mice, and the neuroprotective effect was pronounced across multiple brain regions. The collected data highlights how GENUS-mediated regulation of neuronal activity across the brain region can affect genes and proteins involved in intracellular transport and synaptic function of degenerative neurons. These results establish a connection between gamma vibration by GENUS in the subject, functional binding of neural networks across multiple brain regions, neuroprotection, and ability to perform actions.

In summary, one embodiment of the invention relates to a method of treating a subject in need of treatment for dementia or Alzheimer's disease, the method comprising: A) a plurality of subjects comprising at least the prefrontal cortex (PFC) and hippocampus of the subject And non-invasively delivering a chronic visual stimulus having a frequency of about 30 Hz to about 50 Hz to the subject to tune the synchronized gamma vibration in the brain region of the.

Another embodiment of the invention relates to a method of treating dementia or Alzheimer's disease in a subject in need thereof, the method comprising: A) synchronizing synchronized gamma vibrations in multiple brain regions of the subject and multiple And non-invasively delivering a chronic visual stimulus having a frequency of about 30 Hz to about 50 Hz to the subject to improve abnormally modified genes and proteins in neurons regressing in the brain region.

Another embodiment of the invention relates to a method of treating a subject in need of treatment for dementia or Alzheimer's disease, the method comprising: A) a frequency between 30 Hz and 50 Hz between multiple brain regions of the subject Non-invasively delivering a chronic visual stimulus having a frequency of about 30 Hz to about 50 Hz to the subject to significantly increase gamma interference, thereby simultaneously synchronizing synchronized gamma vibrations in multiple brain regions of the subject.

It should be understood that all combinations of the above and further concepts discussed in more detail below (if these concepts are not mutually inconsistent) are contemplated as part of the subject matter of the invention disclosed herein. In particular, all combinations of claimed subject matter appearing at the end of the disclosure are considered to be part of the subject matter of the invention disclosed herein. It should also be understood that, as a term that may appear in any disclosure incorporated herein by reference, terms explicitly used herein should be given the meaning that most closely matches the particular concept disclosed herein.

Other systems, processes and features will become apparent to those skilled in the art upon review of the following drawings and detailed description. All such additional systems, processes and features are intended to be included within this description, within the scope of the invention, and protected by the appended claims.

The patent or application file contains at least one drawing executed in color.
Those skilled in the art will understand that the drawings are primarily for illustrative purposes and are not intended to limit the scope of the subject matter of the invention described herein. The drawings are not necessarily to scale; In some cases, various aspects of the subject matter disclosed herein may be exaggerated or enlarged in the drawings to facilitate understanding of different features. In the drawings, like reference numbers generally indicate, for example, similar features (eg, functionally similar elements and/or structurally similar elements).
1A-1J show that, according to the disclosed invention, visual stimulation synchronizes gamma vibrations in multiple brain regions of a subject past the visual cortex.
2A-2F show, according to the disclosed invention, chronic 40 Hz (but not 80 Hz) visual blink stimulation reduces amyloid plaques beyond the subject's visual cortex.
3A-3J show that in accordance with the disclosed invention, chronic visual stimulation improves Alzheimer's disease related pathology and particularly reduces or prevents neurodegeneration in subjects.
4A-4Q show that, according to the disclosed invention, chronic visual stimulation reduces inflammatory responses in subjects' microglia.
5A-5I show, according to the disclosed invention, chronic visual stimulation alters synaptic function and intracellular transport in neurons.
6A-6I show that, according to the disclosed invention, chronic visual stimulation alters behavior in a multi-object model of Alzheimer's disease.
7A-I show that, according to the disclosed invention, chronic visual stimulation tunes gamma vibrations beyond the visual cortex in a neurodegenerative mouse model.
8A-8I show that, according to the disclosed invention, chronic visual stimulation reduces AD related pathology in 5XFAD mice beyond the visual cortex.
9A-9G, according to the disclosed invention, chronic visual stimulation improves AD related pathology in P301S and CK-p25 mice.
10A-10N show that, according to the disclosed invention, chronic visual stimulation transforms microglia and enhances intracellular transport and synaptic transmission in neurons.
11A-11J show behavioral properties of effects of acute and chronic visual stimuli on a subject, in accordance with the disclosed invention.
12A-12C show that, according to the disclosed invention, chronic visual stimulation at 80 Hz did not affect the Morris underwater maze in 5XFAD mice.

The following is a more detailed description of various concepts related to systems and methods for preventing, alleviating and/or treating dementia through visual stimuli that bind to higher-order brain regions, reduce neurodegeneration and neuroinflammation, and improve cognitive function. And implementations of the systems and methods. It should be understood that the various concepts introduced above and discussed in more detail below can be implemented in various ways. Examples of specific embodiments and applications are primarily provided for illustrative purposes so that those skilled in the art can implement the embodiments and alternatives apparent to those skilled in the art.

The drawings and example implementations described below are not intended to limit the scope of this implementation to a single implementation. Other implementations are possible by exchanging some or all of the described or illustrated elements. In addition, where certain elements of the disclosed exemplary embodiments can be partially or fully implemented using known components, in some cases only those portions of these known components required to understand the present embodiment are described, Detailed descriptions of other parts of these known components are omitted so as not to obscure the present embodiment.

In this disclosure, we describe Alzheimer's disease, a technique of the invention that involves the treatment of a subject with chronic non-invasive visual stimulation, referred to herein as "Gamma ENtrainment Using Sensory visual stimuli" (GENUS). Dealing with symptoms related to dementia, including (AD), and demonstrating that it affects AD pathology in the brain area beyond the visual cortex. In an exemplary embodiment, we validated the effect of chronic visual GENUS in multiple mouse models of neurodegeneration, including Tau P301S, CK-p25, and reminiscent 5XFAD mice. We have observed that chronic visual GENUS tunes gamma vibration in multiple brain regions (including higher-order brain regions) and induces functional binding at low gamma frequencies across these brain regions. Throughout the multiple neurodegenerative disease mouse models we tested, we improved neuronal and synaptic density in multiple higher-order brain regions, as chronic visual GENUS improved a number of AD-related pathologies including amyloid plaques, tau hyperphosphorylation and brain atrophy. It has been found to prevent loss. Transcriptional and proteomic profiling improves or repairs genes and proteins involved in abnormally modified membrane transport, intracellular transport and synaptic function in degenerating neurons of P301S and CK-p25 mice, and is repaired by chronic GENUS, microglia It has been demonstrated that my immune response is reduced. In view of this wide range of neuroprotective effects, we have further investigated the effect of chronic daily GENUS on cognitive function and demonstrate improved performance of behavioral tasks in multiple AD mouse models. Taken together, our results highlight the neuroprotective efficacy of visual GENUS in treating subjects for dementia, including AD.

As detailed below, several studies were conducted to evaluate the effect of chronic visual stimulation on multiple brain regions. The visual stimulus generally had a frequency of about 30 Hz to 50 Hz, and particular attention was paid to the frequency of 40 Hz or about 40 Hz for visual stimulation. In some embodiments, a light emitting diode (LED) based device was used to deliver the starting stimulus, and the LED based device was driven with a square wave current pattern with a 50% duty cycle. However, it should be understood that various types of devices (other than LED-based devices) can be used to effectively deliver visual stimuli at various frequencies within the ranges mentioned herein. In addition, a duty cycle other than 50%, as well as waveforms other than square wave forms, can be used to effectively generate visual stimuli at various frequencies within the ranges mentioned herein.

In addition, various treatment protocols described herein, including subject exposure time for 1 hour of visual stimulation per day, and subject exposure time for 7 days, 22 days (about 3 weeks) and 42 days (about 6 weeks), are described herein. Was used in However, it should be understood that other subject exposure times and subject exposure durations can be used to deliver visual stimuli at various frequencies within the ranges referred to herein and to effectively treat dementia, including Alzheimer's disease. For example, exposure times greater than 1 hour per day (e.g., delivered multiple times in 1 hour increments, delivered in shorter units, or delivered in increments in longer units), and/or less than 3 weeks, 3 weeks Between 6 and 6 weeks, exposure periods of more than 6 weeks can be used in different combinations and permutations to effectively treat dementia, including Alzheimer's disease.

In the following, we first provide a summary of the experiments and each observation, followed by further details of the experimental protocols to show the efficacy of visual GENUS for treatment of dementia subjects, including Alzheimer's disease.

Visual genus affects higher-order brain regions

We first performed c-Fos immunohistochemical staining as a marker of neuronal activity in wild-type C57Bl/6J mice, so that a 40 Hz visual stimulus can pass through the visual cortex and modulate neuronal activity in the brain area, or it can be transferred to the visual cortex. To determine if it is limited (Fig. 1a). A visual stimulus was delivered at a specific frequency with a 50% duty cycle using a custom LED device (e.g. lit for 12.5 ms and turned off for 12.5 ms for a 40 Hz stimulus) (Iaccarino et al., 2016; Singer et al., in press). Mice were accustomed to their environment for 3 days and then exposed to 1 hour of 40 Hz visual stimulation. They were then returned to their home cage 1 hour prior to sacrifice, brain fragments were collected, sectioned and labeled with c-Fos antibody. We found that a 40 Hz visual stimulus showed a significant increase in c-Fos positive neurons in the visual cortex (V1) (FIGS. 1A-1B ), and also in the somatosensory cortex (SS1), hippocampal region CA1, and prefrontal cortex subjects. A significant increase in cortical (CC) regions was noted (FIGS. 1A-1B ).

To understand how 40 Hz visual stimulation in these brain regions can change ongoing neuronal activity, we implanted a custom microdrive (tungsten wire electrode; see method for details) into a C57Bl/6J mouse, acting freely Local field translocation (LFP) was simultaneously recorded from the target regions of V1, SS1, CA1, and prefrontal cortex (PFC) of the mouse. To reduce navigational behavior, mice were confined in a small GENUS box (8 x 8 inches) and V1, SS1, CA1, and prefrontal cortex when exposed to unblocked 40 Hz visual stimuli initially blocked for 10 minutes each Local field potential (LFP) was simultaneously recorded from (PFC) (FIGS. 1A and 7A ). Acute 40 Hz visual stimulation significantly increased the output of low gamma vibrations of about 35-45 Hz, with a peak frequency of 40 Hz at V1 (Figures 1C-1D) and a small but significant increase in CA1 (Figures 1C-1D), This is consistent with previous findings in head-restrained mice (Iaccarino et al., 2016). A small but significant increase in gamma output was also observed in SS1 and PFC (FIGS. 1C-1D), consistent with the increase in c-Fos positive cells we observed across these regions (FIG. 1B).

To assess whether these visual stimulation-induced vibrations could accompany neuronal excitation downstream of the brain region of the visual cortex, we implanted a quadrangular tube targeted to the hippocampal region CA1 into a cohort of C57Bl/6J mice to record single unit activity. Did. Under the light-blocked reference conditions, CA1 vertebral cells showed strong phase immobilization at low gamma (about 35-45 Hz) with preferential discharge at peak, consistent with previous reports (Bragin et al., 1995; Middleton and McHugh , 2016) (Figure 1e). While not changing the preferred phase, visual stimulation at 40 Hz quantifies the increased average result length across the population (Figure 1F), and stronger phase fixation of individual neurons (MRL; blocked stimulation and visible visual stimulation at 40 Hz) P = 0.01), indicating that gamma frequency visual stimulation acts to drive a population of hippocampal neurons in a more temporally organized manner.

To test the applicability of GENUS to impaired neuron systems, several mouse models of neurodegeneration were used to verify whether gamma vibration could be induced by visual stimulation, as observed in C57Bl/6J animals. We used the CK-p25 mouse strain, in which expression of the Cdk5 activator p25 is driven in a manner inducible by the excitatory neuron-specific CaMKIIα promoter (CaMKIIα promoter- tTA x TetO- p25+GFP) (Cruz et al., 2003). ). After recovering doxycycline from the food, CK-p25 shows progressive neurons and synaptic loss with severe cognitive impairment by 6 weeks of p25 induction (Cruz et al., 2003). Consistent with these results 6 weeks after induction, in vivo LFP recordings from CK-p25 mice showed significantly reduced gamma spectral output at 35-45 Hz compared to control mice in V1, CA1, and PFC (FIG. 7B ). -7c). Despite these changes, 40 Hz visual stimulation could still significantly improve gamma vibration in V1, CA1, and PFC (FIG. 7C ). We further investigated Tau P301S mice expressing high levels of humanized mutant microtubule-associated protein tau and having tau aggregates associated with frontotemporal dementia at the early age of 5 months (Yoshiyama et al., 2007). Eight-month-old P301S mice showed improved gamma output at 40 Hz visual stimulation in V1 and PFC at ages with synaptic and neuronal loss and cognitive defects (FIGS. 7D-7E ). These results indicate that GENUS is sufficient to accompany gamma vibration in AD mice regardless of the specific mode of neuronal damage in these mice.

To ensure that multiple exposures of visual stimulation can still synchronize gamma vibrations even after prolonged periods, we used C57Bl/6J and p25 induced CK-p25 mice exposed to 40 Hz visual stimulation for 42 days (1 hour/day). The GENUS protocol was used (Fig. 7F, Fig. 7H). On day 43, LFP recorded from V1, SS1, CA1, and PFC was consistent with single trial acute GENUS application and showed significantly enhanced 40 Hz gamma output across all regions of C57Bl/6J mice (FIG. 7F ). In a similar manner, CK-p25 mice on day 43 (currently 85 days after p25 induction) also showed increased low gamma output in V1, CA1, and PFC (FIG. 7H ). These results, along with c-Fos immunostaining data, show that acute and chronic 40 Hz visual stimulation reinforces not only the visual cortex, but also other higher order cortex including CA1, SS1 and PFC.

Since 40 Hz visual stimulation (both acute and chronic) simultaneously enhances 40 Hz output across V1, SS1, CA1 and PFC, we act to enable GENUS to regulate neuronal activity between the visual cortex and these other higher-order brain structures. It was also proven to do. We calculated the interference across these brain regions during 40 Hz visual stimulation with blocked and unblocked light using the Weighted Phase Delay Index (WPLI) method, which minimizes potential contamination through volume conduction. (Vinck et al., 2011). LFP of C57Bl/6J mice was analyzed across electrode pairs located in V1 to CA1, V1 to SS1, V1 to PFC, CA1 to SS1 and CA1 to PFC. The average rate was not significantly different in the duration during the 40 Hz visual stimulation with or without light blocking, and any potential differences in motility activity were ignored. Acute 40 Hz visual stimulation significantly increased 30-50 Hz low gamma interference in the visual cortex or specifically the brain region irradiated between V1-CA1, V1-SS1 & V1-PFC (Figures 1g, 1h).

To assess the long-term effects of chronic GENUS on neuronal oscillation coordinated over a wider brain structure, we performed C57Bl/6J and p25 induced CK-p25 mice exposed to visual stimulation for 1 hour/day for 42 hours. The WPLI analysis was applied to LFP collected from V1, SS1, CA1, and PFC at. We compared the light blocking conditions with a significant increase of 30-50 Hz low gamma WPLI not only between V1~SS1, V1~PFC, and CA1~PFCV1 (Figure 7g), but also between V1 and CA1 during 40 Hz visual stimulation. Found (Fig. 7g). Likewise, after chronic GENUS, CK-p25 mice also showed a significant increase of 30-50 Hz low gamma WPLI between V1-CA1, CA1 -PFC and V1 -PFC compared to light blocking conditions (Figure 7i). Overall, these data suggest that a 40 Hz visual stimulus enhances neuronal vibrational activity (30-50 Hz low gamma WPLI) coordinated in several higher order cortex of mice, not only between regions but also locally (40 Hz tuning). However, it is important to demonstrate that the changes we have observed are specific to low gamma frequency stimuli. To test this, we exposed the C57Bl/6J mice to an 80 Hz visual stimulus and delivered with a 50% duty cycle to ensure that the mice receive similar light intensity and duration of exposure as in our 40 Hz stimulation experiment (FIG. 1i). No significant change in the 80 Hz spectral output was observed in the visual cortex during the 80 Hz visual stimulation compared to the pre-stimulation period (we observed the largest power increase with 40 Hz GENUS) (FIGS. 1I, 1J).

Chronic genus improves amyloid plaque beyond the visual cortex

Iaccarino et al. (2016) show that acute time GENUS reduces amyloid levels in V1 of young pre-symptomatic 5XFAD mice, while visual GENUS of 7 days in 6-month-old 5XFAD mice with more advanced pathology not only reduces amyloid levels. , Amyloid plaques were also demonstrated to improve. We therefore aimed to investigate whether 7 days of 40 Hz visual stimulation in V1 of 11 month old 5XFAD mice (as well as CA1, SS1, and PFC) could affect amyloid plaque pathology. Towards this goal, we place the mice in a GENUS stimulation cage (Fig. 8A) and receive them a 40 Hz or 80 Hz visual stimulus (transmitting an amount of light similar to 40 Hz) for 1 hour per day for 7 days. However, the gamma vibration was not tuned at V1 (FIGS. 1I, 1J), and the amyloid plaque load was examined (FIG. 2A). After 7 days of 40 Hz visual stimulation, we saw a decrease in amyloid plaques in the visual cortex (FIGS. 2A and 2B ), but no differences were found in SS1 or CA1 compared to unstimulated mice (FIGS. 2A and 2B ). . According to the LFP analysis, 5 HzFAD mice subjected to 80 Hz visual stimulation showed no change in amyloid plaque load in V1 or CA1, and showed a moderate increase in SS1 (FIGS. 2A and 2B ). These results show that reduction of amyloid plaques after 7 days of visual GENUS is limited to V1 and specific for 40 Hz visual stimulation.

We then started at 9 months of age with 5XFAD mice, extended the GENUS protocol to 22 days, and quantified amyloid plaques when mice were about 10 months of age (Figure 2C). We found that GENUS on day 22 significantly reduced the extent and number of amyloid plaques in V1 (Figures 2c, 2d and 8b-8d), which was 6 months old 5XFAD (Iaccarino et al., 2016) and 11 months old 5XFAD mice In agreement with the reduction of amyloid plaques after 7 days of GENUS (FIGS. 2A, 2B). Importantly, GENUS on day 22 is also sufficient to reduce the degree and number of plaques in the SS1, CA1 and CC regions of the prefrontal cortex (Figures 2D, 8B-8D). This effect was again specific for 40 Hz stimulation, as 80 Hz stimulation at 22 days did not alter amyloid plaques in V1, SS1, or CA1 of 5XFAD mice when compared to unstimulated 5XFAD mice (Figures 2e, 2f). .

We chose to apply long-term GENUS starting at 9 months of age in 5XFAD mice, as previous studies showed that gradual loss of neurons and synaptic markers in the 5XFAD model began at the age of about 9 months (Oakley et al., 2006). . According to this study, we observed a significant decrease in the number of neurons in both CA1 and CC of 10 month old 5XFAD mice, compared to wild-type litter pups of the same age (Figures 8C, 8E). In contrast, 5XFAD mice receiving GENUS on day 22 showed significantly reduced neuronal loss compared to unstimulated mice, in addition to reduced amyloid load (FIGS. 2C, 2D and FIGS. 8B-8D) (FIG. 8E ). . Similarly, we observed significant loss of bassoon (puncta) spots in CA1 and CC of unstimulated 5XAFD mice, whereas chronic GENUS for 22 days reduced synaptic loss (Figure 8f, 8g). Reduced neurodegeneration and amyloid pathology after chronic GENUS were not the result of altered APP metastasis gene expression since we found no difference in the expression of full-length APP protein in mice receiving GENUS compared to the unstimulated control group (FIG. 8H ). , 8i). These results demonstrate that chronic visual GENUS causes decreased amyloid plaque load, and reduced loss of neurons and synapses, in several brain regions of 5XFAD mice.

Chronic genus reduces neurodegeneration

To further explore the possibility of chronic GENUS affecting disease pathology and reducing neuronal loss, as can be seen in 5XFAD mice (Figures 8C, 8E), we subsequently followed the neurodegenerative Tau P301S and CK-p25 mice. The model was examined. At the age of 8 months, Tau P301S mice show significant neuropathology (Yoshiyama et al., 2007). Therefore, we took a cohort of Tau P301S mice and allowed them to receive GENUS for 1 hour per day for 7 days from 7 months of age, irritated or irradiated, and their Tau phosphorylation levels and Tau related pathologies at about 8 months of age were examined ( 3A to 3E, and FIGS. 9A and 9B). We observed higher phosphorylation of tau at the S202/T205 residues in V1, SS1, CA1 and CC of Tau P301S mice compared to the wild-type naive litter (Figure 3A, 3B). However, Tau P301S mice receiving chronic GENUS significantly reduced S202/T205 tau phosphorylation compared to unstimulated P301S mice (FIGS. 3A and 3B ). In AD and P301S mice, Tau protein was over-phosphorylated at multiple residues (Hanger y et al., 2007; Wang et al., 2013; Foidl and Humpel, 2018; Kimura et al., 2018), and thus the unbiased Ser/Thr ( The S/T) phosphoprotein physics approach examines the extent to which GENUS stimulation can affect tau phosphorylation. We identified 46 superphosphorylated S/T residues and dephosphorylated single residues (S451) in Tau P301S mice compared to WT naive litter pups (FIG. 3C ). Our analysis also revealed that chronic GENUS reduced phosphorylation in Tau protein at 6 S/T sites and increased phosphorylation at S451, indicating that GENUS affects Tau phosphorylation at multiple sites (Figure 3c). ).

We next characterize neuronal loss in Tau P301S mice, and as previously reported, they show a significant decrease in neuron counts in V1, CA1, SS1, and CC as quantified by the number of NeuN positive cells. (Fig. 3d, 3e). Tau P301S mice that received GENUS from the age of 7 months for 22 days (at the beginning of neuronal loss) showed significantly reduced neuronal grooming in all brain regions we investigated compared to the unstimulated control group (FIG. 3D, 3e). We next examined brain weight and lateral ventricular size, and no difference was observed between WT unstimulated and GENUS stimulated P301S mice (FIG. 9B ).

We then switched to a CK-p25 model showing AD-like pathological features, such as brain atrophy, cortical contraction, and abnormal ventricular dilatation after 6 weeks of p25 induction (Cruz et al., 2003). To investigate the extent to which chronic GENUS can improve this abnormality, we simultaneously induced p25 in CK-p25 mice for 6 weeks while also exposing them to 1 hour of GENUS daily (FIG. 3F). Unstimulated CK-p25 mice showed significantly reduced brain weight and cortical thickness compared to CK Naive litter pups (CaMKIIα promoter- tTA; Cruz et al., 2003) (Figure 3g). Chronic GENUS significantly reduced cortical thinning during p25 induction compared to unstimulated CK-p25 mice, but did not significantly alter brain weight (Figures 3G, and Figure 9D). In both human AD and CK-p25 transgenic models, cortical contraction was closely associated with ventricular dilatation (Cruz et al., 2003), and we also dramatically reduced ventricular dilatation in CK-p25 mice receiving chronic GENUS. Was observed (FIGS. 3H and 9E ). In addition, CK-p25 mice treated with chronic GENUS during p25 induction also significantly reduced neuronal loss in the V1, SS1, CA1, and CC regions of PFC compared to the unstimulated control group (Figures 3i, 3j). It has been reported that DNA damage in the form of double-strand break (DBS) indicates an early marker for neurodegeneration in CK-p25 mice (Kim et al., 2008). According to these results and the GENUS-mediated reduction in neuronal death, we also significantly reduced, as analyzed by the number of γH2AX positive cells, a well-established marker of DSB in V1, SS1, and CA1. It was observed (Fig. 9g). Because total tau and p25 expression (in P301S and CK-p25 mice, respectively) did not differ between GENUS and unstimulated groups, this neuroprotective effect of chronic GENUS was not induced to alter transplant gene expression in mutants ( 9A, 9C, 9F). Taken together, these observations establish that chronic GENUS is neuroprotective in P301S and CK-p25 mouse models with severe neurodegeneration.

Chronic genus reduces inflammatory response

We also demonstrated that the reduced neurodegeneration observed in Tau P301S and CK-p25 mice after chronic GENUS can be partially mediated by favorable microglia responses. We performed unbiased RNA sequencing in the visual cortex of unstimulated P301S and CK-p25 mice and their respective WT controls, as well as P301S tau and CK-p25 mice that received GENUS stimulation for 22 and 42 days (FIG. 4A ). . The visual cortex was dissected, digested by enzymes, and microglia were identified by CD11b and CD45 immunostaining, and then isolated using a fluorescence activated cell sorter (FACS) as described above (Mathys et al., 2017). (FIGS. 10A, 10B). We isolated 35,000 microglia from each mouse, extracted total RNA separately from each mouse, and checked the quality of RNA before RNA-seq. Averaged 28.69 million readings per sample, of which 89.42% were sorted.

Compared to CK naive mice, RNA sequencing revealed that microglia derived from unstimulated CK-p25 mice had 2333 upregulated genes (FIG. 4A ). We next performed gene ontology (GO) analysis, and observed that upregulated genes are involved in protein synthesis, ribosome regulation, and immune responses (including viral immune response, antigen presentation and immune response regulation). , This was consistent with previous reports (Mathys et al., 2017) (Figure 4b). In comparison, the identified 2019 down-regulated genes were primarily associated with cell migration, cell morphogenesis and vasculature development (FIG. 4B ). After chronic GENUS, 355 genes were upregulated in CK-p25 mice (compared to unstimulated CK-p25), while these genes were found to be involved in protein synthesis, mitotic cell cycle regulation, membrane transport and vesicle-mediated transport, The 515 down-regulated genes were mostly associated with GTPase activity, proteolysis and immune responses (including MHC-1 mediated antigen treatment presentation and immuno-adaptability) (FIG. 4C ). These results indicate that chronic GENUS significantly affects microglia function in CK-p25 mice, alleviates inflammation and makes phagocytosis, migration and protein degradation as much as possible.

In unstimulated Tau P301S mice, we found a total of 331 up-regulated genes and 292 down-regulated genes compared to WT naive litters (FIG. 10C ). While gene ontology (GO) analysis correlated up-regulated genes with protein synthesis and inflammatory/immune responses, down-regulated genes were more associated with cytoskeletal tissue, cell migration and brain development (Figure 10D). Microglial cells obtained from Tau P301S mice after chronic GENUS (22 days) showed 238 up-regulated genes and 244 down-regulated genes compared to unstimulated Tau P301S mice. Here, the up-regulated gene is associated with membrane transport using down-regulated genes involved in cellular catalytic proteolysis, cell migration, cell morphogenesis and gene expression, translation initiation and interferon responses (FIGS. 10C and 10D ). . Therefore, CK-p25 mice treated with chronic GENUS showed significantly similar transcript changes to Tau P301S mice treated with chronic GENUS. Taken together, our microglia-specific transcriptome analysis shows that chronic GENUS independently morphologically modifies microglia, enhances protein degradation, and enhances microglia, independent of the specific transgenic model (P301S or CK-p25). It acts to reduce the mediated immune response, creating a disease state.

To further demonstrate this finding, we used CK-p25 and Tau P301S mice after chronic GENUS (CK/p25: 1 hour/day for 42 days to induce p25; Tau P301S: 22 days), as well as each unstimulated And immunohistochemical staining using brain slices of the naive control. The CK-p25 microglia response is described above, and the initial response is characterized by increased proliferation and late response marked by elevation of the MHC class II and interferon pathways (Mathys et al., 2017). We first used microglia specific marker Iba1 to perform immunohistochemistry and three-dimensional rendering, and revealed an increase in microglia number and extensive changes in morphology in V1 of CK-p25 mice induced for 6 weeks (Figure 4d). ~ 4i and Figure 10e). The CK-p25 animal microglia did not show a significant difference in cell soma volume as a whole compared to the control group (FIG. 4f ), but the majority were more complex'bushy' dendritic cells associated with axonal and terminal synaptic degeneration. The pattern (arrow head) (Fig. 4d; lower center panel) was shown (Jensen et al., 1994; Jorgensen et al., 1993). We also have a phenotype known to exist after dispersing brain damage in human subjects, a large portion of microglia with a long, elongated rod-like body (arrows) (Fig. 4d, 4i), traumatic brain injury, and human subjects (Bachstetter et al., 2017; Taylor et al., 2014). In addition, despite the reduced projection volume (Figure 4g), CK-p25 microglia were more physically close to each other than the CK Naive control, as analyzed by measuring the minimum distance between microglia (Figure 4d, 4h). ). This represents their zone loss and contrasts with the resting state, with each microglial cell having its own occupied area, which generally rarely overlaps between adjacent zones (Del Rio-Hortega, 1932; Nimmerjahn et al., 2005).

Chronic GENUS significantly reduced the number of microglia in CK-p25 mice compared to unstimulated mice, but remained higher than in naive CK mice (FIGS. 4D and 4E ). After chronic GENUS, the total volume of microglia protuberances was such that there was little contraction and there was no significant difference compared to the CK-p25 and CK naive groups (Fig. 4g). Importantly, the minimum distance between microglia after chronic GENUS was comparable to that of CK naive animals (Figures 4D, 4H), suggesting preservation of microglia zones with chronic GENUS. Finally, the increase in the overall volume of rod-shaped microglia in CK-p25 animals treated with chronic GENUS was significantly reduced, but remained significantly higher than in the CK naive group (FIG. 4i ). We next characterized Tau P301S microglia and found that P301S tends to increase microglia, but did not reach statistical significance in our Iba1 immunostaining (Figures 4k, 4l). In addition, there was no difference in the total volume of microglial soma in P301S compared to WT naive mice (Fig. 4k, 4m). However, the total volume of microglial processes in Tau P301S mice after chronic GENUS was significantly different from that of unstimulated P301S mice, and was similar to that of WT naive litter pups (FIGS. 4K and 4N ). These results highlight the range of microglia in baseline and disease conditions, and overall indicate the effect of chronic GENUS to alleviate disease-related microglia morphology in P301S and CK-p25 mice.

We next examined the GO term for an immune response that appeared to be affected by chronic GENUS. Immunohistochemistry using an antibody specific for the interferon-reactive gene CD40 showed a significant increase in CK-p25 compared to CK naive mice (Mathys et al., 2017) (Fig. 4d, 4j). Chronic GENUS significantly reduced the CD40 signal intensity in CK-p25 mice, but remained significantly higher than in the CK naive control (FIGS. 4D, 4J). We next investigated another immune response gene, C1q (classical complement component), which was significantly upregulated in microglia-specific RNA-seq experiments and was previously associated with synaptic loss in AD mouse models (Hong et al., 2016). Immunohistochemistry for C1q showed elevated signal in V1 of unstimulated CK-p25 and Tau P301S mice compared to their respective naive control litters (FIGS. 4o, 4p, 4q). Chronic GENUS significantly decreased the increase in C1q of CK-p25 mice, but the C1q intensity remained higher than the CK naive level (FIGS. 4o and 4p ). In P301S mice, chronic GENUS attenuated the increase in C1q, so there was no significant difference between WT naive and unstimulated P301S mice (FIGS. 4o, 4q). Overall, our immune histochemistry results consistently show that chronic GENUS helps reduce the immune response of microglia.

Chronic genus alters the regulation of synaptic transmission and intracellular transport in neurons

We next isolated NeuN positive (NeuN+) neuronuclei and performed an unbiased RNA-seq analysis to study the effect of chronic GENUS on gene expression in neurons in V1 of CK-p25 mice and Tau P301S (FIG. 5A). , 5b and 10f to 10g). Visual cortex was harvested from mice after chronic GENUS (CK-p25: p25 induction at 42 days; Tau P301S: 22 days). Thereafter, 100,000 neuronuclei were placed in lysis buffer, sorted by FACS, and then RNA was extracted and sequenced (FIGS. 5A and 5B). According to the results of studies on brain weight and quantification of NeuN and cortical thickness reduction using immunohistochemistry (FIGS. 3G, 3I and 9D), we have no irritation CK when compared to naive CK litter (100 ± 3.87). It was found to significantly reduce the percentage of NeuN+ nuclei in the -p25 mice (81.56 ± 3.29), which was not evident in CK-p25 mice treated with chronic GENUS (88.56 ± 4.49) (Figures 10f, 10h). Similarly, the percentage of NeuN+ nuclei in unstimulated Tau P301S mice was significantly lower than that of naive WT litter pups (100 ± 3.87) (86.11 ± 2.26), but Tau P301S mice treated with chronic GENUS were consistent with our immunohistochemistry findings. However, it was not different from WT mice (96.05±3.99) (FIGS. 10G, 10H) (FIGS. 3E, 3J). These results reduce the loss of nerve nuclei with chronic GENUS in both the CK-p25 and Tau P301S neurodegenerative mouse models and indicate the overall neuroprotective effect of GENUS.

We next performed RNA-seq from neuronal RNA sorted by this FACS. Averaged 18.09 million and 22.79 million readings per sample, of which 85.23% and 84.45% were sorted from CK-p25 and P301S mice, respectively. Non-biased transcriptional analysis of the NeuN+ nucleus showed up-regulated genes in CK-p25 (618 genes) and Tau P301S (351 genes) compared to each control mouse (CK-p25: 565 genes; Tau P301S: 229 Dog genes), and showed that more genes were down-regulated (FIGS. 5A and 5B ). Chronic GENUS showed similar numbers of up-regulated vs. down-regulated genes in CK-p25 (409 up; 422 down) and Tau P301S (220 up; 221 down) compared to each non-stimulated control. Result (FIGS. 5A, 5B).

We then performed GO analysis to investigate the biological function associated with the differently expressed genes. Neuron-specific down-regulated genes (618 genes) in CK-p25 mice after chronic GENUS include chemical synaptic transmission, intracellular transport, autophagy, ATP metabolic processes, trans-synaptic signal transduction, and cell-cell signaling Involved in delivery (FIG. 5A ). Interestingly, GENUS rescued this biological process by significantly upregulating the genes involved in these processes in CK-p25 mice (FIG. 5A; right panel). Down-regulated genes in Tau P301S (351 genes) were involved in the regulation of intracellular transport including chemical synaptic transmission, trans-synaptic signal transduction, vesicle-mediated transport, midbrain development, and apoptosis (FIG. 5B). These same processes (including regulation of vesicle-mediated transport, intracellular transport, synaptic transmission, midbrain development, and apoptosis processes) all upstream after chronic GENUS in Tau P301S mice, as revealed from the upper biological functions associated with the upregulated genes. Were adjusted (FIGS. 5B and 10I ). On the other hand, some biological processes are up (compared to the naive control) in both CK-p25 and Tau P301S, including those involved in DNA double strand cleavage and DSB repair, according to our immunohistochemistry data for γH2AX. Adjusted (Figure 9g). Importantly, genes that were down-regulated in CK-p25 and Tau P301S mice after chronic GENUS included those known to be essential for the apoptosis pathway in response to DNA damage, consistent with the shift of neurons toward less degeneration. Taken together, these data show that in the two models of mechanically distinct neurodegenerative mice (CK-p25 and Tau P301S), cells with similar altered patterns of gene regulation and downregulation of (synaptic function, intracellular transport and apoptosis regulation) Converging biological functions, which ultimately promotes the disappearance of neurons. Importantly, many genes involved in relieving defects in intracellular transport, including synaptic transmission, synaptic tissue and vesicle-mediated transport after chronic GENUS are upregulated (FIGS. 5A, 5B and 10H).

To further characterize and verify this biological process altered by chronic GENUS, we performed unbiased liquid chromatography-tandem mass spectrometry (LC-MS/MS) to determine the CK-p25 and Tau P301S mice. Differential expression of protein and S/T phosphorylation at V1 were investigated (Fig. 5C, 5D). We first identified by mass spectrometry in the combined CK-p25 group (CK naive control, and CK-p25 with or without GENUS) and in the combined Tau P301S group (WT naive control, P301S with or without GENUS), respectively All proteins compared to all RNAs detected from neuron-specific RNA-seq data were examined. We found that 92.75% and 91.95% of the total proteins identified in the combined CK-p25 and combined Tau P301S groups, respectively, were mapped to genes expressed in neuron specific RNA-seq data (FIGS. 5C, 5D). , This suggests that most of the identified proteins are involved in neuronal function. We next compared S/T phosphorylated proteins differently in CK-p25 and Tau P301S mice to their respective naïve control litters and overall increased S/T-phosphorylated protein in both CK-p25 and Tau P301S mice. (FIGS. 5C, 5D; lower left panel), indicating abnormal modification of the functional protein in both neurodegenerative mouse models. Chronic GENUS reduced S/T phosphorylation of protein compared to each non-stimulated control in CK-p25 and Tau P301S mice (Figure 5c, 5d; bottom right panel). Consistent with our neuron specific gene expression analysis (FIGS. 5A, 5B), chronic GENUS delivers chemical synaptic transmission, dendrite development, long-term potentiation, regulation of vesicle-mediated transport, vesicles within synapses. The proteins involved in the regulation of mediated transport and intracellular transport have been modified (FIGS. 5E and 10J-10M ), indicating that this process is modified in degenerative neurons and improved with chronic GENUS.

One exemplary protein from our human proteomics analysis associated with vesicle transport, endocytosis, and synaptic transport is Dynamine 1 (DNM-1) (Armbruster et al., 2013), which is found in both CK-p25 and Tau P301S. It is associated with several GO terms in the annotation of differently S/T-phosphorylated proteins after chronic GENUS (FIG. 5E ). Phosphorylation regulation in Ser774 is required for endocytosis of synaptic vesicles (Clayton et al., 2009) and is one of many residues found to be hyperphosphorylated and reduced to chronic GENUS in CK-p25 and Tau P301S mice. We performed immunohistochemistry and western blotting, and found that DMN-1 Ser774 phosphorylation was significantly increased in CK-p25 and Tau P301 mice and decreased to chronic GENUS (Figures 5f and 10m, 10n). We have also performed immunohistochemistry for vesicular and glutamate transporter 1 (vGlut1), another protein related to vesicle and neurotransmitter transport, synaptic transport, and learning and memory (Balschun et al., 2009). We have vGlut1 spots in the CK-p25 mice after chronic GNEUS, as well as in the SS1, CA1, and ACC regions, as well as in the V1 of PFC, with significantly higher vGlut1 expression (compared to the unstimulated control), CK naive mice (Figures 5g, 5h) ), it was found to be significantly reduced in CK-p25 mice (Fig. 5g, 5h). Likewise, after chronic GENUS in Tau P301S mice, reduced expression of synaptic marker vGlut1 spots in Tau P301S mice was reduced across these brain regions (FIG. 5i ). These data are consistent with reduced neurons (Figures 3D and 3I) and synaptic loss (Figures 4O, 4P, 4Q) across these brain regions using chronic GENUS in both neurodegenerative models.

Chronic genus transforms behavioral performance

Our results so far show that GENUS can tune neuronal vibrations far beyond V1, including CA1, SS1 and PFC, and amyloid plaques, tau phosphorylation, synaptic loss in all these brain regions of CK-p25 and Tau P301S mice, And AD related pathologies including neuronal loss. Our RNA-seq and human proteomics data support the ability of chronic GENUS to mitigate some disease-related deficits in synaptic transmission and intracellular transport consistent with conserved synaptic function. Therefore, we asked whether chronic GENUS also improves cognitive function. We first sought to determine whether GENUS triggered a systematic behavioral change that could complicate the interpretation of arbitrary cognitive changes. There was no difference in the amount of motor activity in the distance traveled during or after acute GENUS in C57Bl/6J mice (FIGS. 11A-11C ). In addition, seizure sensitivity and novel object identification by picrotoxin were unchanged (FIGS. 11B-11C ). Animals in all groups chronically receiving GENUS stimulation (57BL6: 7 days; CK-p25: 1 hour/day for 42 days inducing p25; Tau P301S: 22 days) had a similar body weight to the unstimulated control group (FIGS. 11f). Finally, GENUS on day 7 did not affect the stress response marker plasma corticosteroid in C57Bl/6J mice (FIG. 11i ).

To assess the cognitive benefits of GENUS, we induced p25 expression for 6 weeks with chronic stimulation with GENUS in CK-p25 mice, and then focused on learning and memory in the CK-p25 mouse model of AD. In the last week, after exposing the mouse to the open field (OF), a new object recognition (NOR) test was performed (FIG. 6A ). Our results showed that GENUS did not affect anxiety in CK-p25 mice (measured as the time spent in the center of the open field arena (Figures 6A, 6B)) and did not alter plasma corticosteroid levels. No (FIG. 11I), suggesting that chronic GENUS did not affect anxiety or stress in CK-p25 mice. Interestingly, GENUS stimulated CK-p25 mice did not show any difference in the amount of exercise activity, but took significantly more time to explore new objects compared to familiar objects (FIGS. 6A, 6C and 11G), This indicates that chronic GENUS improved novel object recognition in CK-p25 mice compared to unstimulated CK-p25 mice. We next performed the Morris Water Maze (MWM) test for 6 weeks and induced CK-p25 mice for 6 weeks of GENUS. In the MWM test, unstimulated CK-p25 mice exhibiting spatial learning failure compared to CK naive mice are marked with longer wait times to find the platform during training (FIG. 6D ). Non-irritated CK-p25 mice exhibited spatial learning dysfunction as seen in the probe test (24 hours after the last training day) compared to the CK Naive group, with reduced platform location visits and reduced time required in the target quadrant (Fischer et al., 2005). ) (FIG. 6D). This failure was significantly improved by chronic GENUS and was not the result of altered swimming speeds maintained similarly throughout the group and across all training days (FIGS. 6D and 11J ).

In order to establish that this GENUS does not limit the improvement of behavioral performance to a single mechanism underlying cognitive impairment, we have tested several AD model mice to establish what is more generally applicable to AD-related cognitive decline. We caused the 8-month-old Tau P301S mice to experience GENUS (1 hour/day) for 22 days, and at 3 weeks of stimulation, mice were tested with the OF and NOR tasks and compared to the unstimulated group of the same age (FIG. 6E ). . Like CK-p25 mice, GENUS did not change the time spent at the center of the arena in P301S mice (Figures 6E, 6F), but neither plasma corticosteroid levels (Figure 11I), which chronic GENUS did in Tau P301S mice. It suggests that the anxiety-like behavior has not been altered. We subsequently performed a new object recognition test and observed that the WT naive and GNEUS stimulated Tau P301S mice showed higher preference for new objects than the existing objects (FIG. 6G ). Similarly, the novel object preference was higher in the unstimulated Tau P301S mice (Figure 6g).

We next performed MWM tests during the 3rd week GENUS in Tau P301S mice. WT naive and GENUS stimulated Tau P301S mice showed a significantly increased learning curve in MWM training compared to unstimulated Tau P301S mice (FIGS. 6H and 11J ). Finally, we tested the effectiveness of GENUS to improve behavior in the AD amyloid model. We had GENUS stimulated 5XFAD mice (22 days-1 hour/day) undergoing MWM testing during the 3rd week training (Figure 6i). GENUS stimulated 5XFAD mice show a slightly lower learning curve during the first 4 days of MWM training, but in contrast to the significantly impaired unstimulated 5XFAD mice, they are improved over several training assays compared to WT litter pups (FIG. 6i ). And Figure 11j). Finally, using 80 Hz visual stimulation, we investigated whether chronic stimulation with different frequencies can affect behavior. Similar to the in vivo recordings at V1, 5XFAD mice that experienced 80 Hz visual stimulation for 22 days showed no difference in MWM compared to the unstimulated 5XFAD control (at the time of probe to find the platform and target crossing in the probe test). Quantitation) (FIGS. 12A-12C ). Taken together, these results specifically indicate that chronic stimulation at 40 Hz improves behavioral performance in several mouse models of neurodegeneration.

Increasing evidence supports the notion that manipulating neuronal network oscillations can mitigate pathological changes and behavioral performance deficits associated with neurological disorders in place of promising strategies (Cho et al., 2015; Iaccarino et al., 2016; Kastanenka et al., 2017; Martinez-Losa et al., 2018; Verret et al., 2012). Here, we demonstrated that chronic daily tuning of low gamma vibrations through 40 Hz visual stimulation was effective in tuning gamma vibrations, even under advanced neurodegenerative conditions, to reduce neuropathology in multiple brain regions. The neuroprotective effects of chronic GENUS include a decrease in microglia-mediated inflammatory response, which increases the expression of genes and proteins that promote synaptic and intracellular transport in neurons, and improves the ability to perform actions.

Chronic visual GENUS tunes low gamma vibrations across brain regions and increases gamma interference

We previously demonstrated that acute one hour (1 hour) visual stimulation at 40 Hz tuned neuronal activity, vibrating in the gamma frequency range, and reduced AD-related phenotype in young, pre-symptomatic 3-month-old 5XFAD mice (Iaccarino et al., 2016). In the present disclosure, we have a number of parts of the brain (including visual cortex (V1), hippocampus CA1, somatosensory cortex (SS1), and frontotemporal cortex (PFC)), despite severe AD-like pathology and loss of neurons. Throughout, it was demonstrated that GENUS significantly increased the low gamma (about 35-45 Hz) vibration output in CK-p25 and Tau P301S mouse models with neurodegeneration. Motor activity may be a disturbing factor in gamma vibration detection, but we have not found any difference in speed or total distance traveled during the recording session between blocking and visible 40 Hz visual stimuli, which is the low gamma tuning we observed during GENUS. Makes the possibility that the level of activity is not related to the difference. In addition, a single unit record analysis in CA1 shows that GENUS can augment neuronal activity in several brain regions downstream from V1 (see also Martorell and Paulson et al., accompanying submission) and less from uncorrelated noise sources. Measurements of the weighted phase delay index (WPLI), which is a sensitive volume conductivity, show that V1, CA1, SS1, and PFC show enhanced low gamma interference and are more functionally coupled with GENUS. This is important in that'communication by interference (CTC)' is essential for cognitive function (Fries, 2015), and it is not surprising that human AD subjects show defects in interference between cortical regions (Stam et al. , 2009).

Visual GENUS gives extensive neuroprotection in several neurodegenerative mouse models

To determine whether chronic application of GENUS can affect pathology in a wider brain area, we have a basic mode, such as the visual cortex and the targeted part of the hippocampus and prefrontal cortex (PFC), highly affected by AD. It focused on other core structures of the network. We first applied GENUS for 7 days in older 5XFAD mice, and found that, despite Iaccarino et al. (2016), amyloid plaques were significantly reduced in V1, but did not alter amyloid levels in the hippocampus (Figure 2a, 2b).

Therefore, we extended the GENUS therapy for 3 weeks using relatively old 5XFAD mice (10 months old) with severe pathology, and amyloid plaque load as well as V1, as well as other distribution parts of the brain including SS1, hippocampus and PFC. Also shows a significant improvement. We applied this prolonged GENUS therapy at the age of initiating neuronal loss in each of these models for 6 weeks throughout p25 induction in the CK-p25 model, 3 weeks in Tau P301S and 5XFAD mice. We are not limited to V1, but also CA1, SS1, and CC (target part of the prefrontal cortex) across the CK-p25, Tau P301S, and 5XFAD mouse models (superphosphorylated tau, synaptic and neuronal loss, and DNA damage A significant decrease was found in each pathology. The neuroprotective effect of GENUS to prevent neuronal loss is particularly in the CK-p25 mouse model, which typically exhibits dramatic brain atrophy, cortical volume contraction, and corresponding ventricular dilatation associated with human AD when neurons and their elongated projections degenerate. It was obvious. Thus, we demonstrate that GENUS exerts a wide range of neuroprotective effects across multiple mouse models with different pathological features.

Vision GENUS alters synaptic function, intracellular transport, and behavior

To investigate the impact of GENUS at the molecular level, we next switched to unbiased transcriptometic and proteomic analysis. Our data from isolated/purified Tau P301S and CK-p25 neurons is consistent with the altered neuron excitation and excitation/inhibition balance previously reported in these neurodegenerative models, and several genes, proteins, and translations that regulate synaptic function. Post-modified protein demonstrated a significant dysregulation (Fischer et al., 2005; Yoshiyama et al., 2007). AD is also known to reduce dendritic branch density, and increasing dendritic density in AD mice by genetic manipulation, pharmacological inhibition of histone deacetylase (HDAC), or photogenetics alleviates cognitive impairment (Fischer et al., 2007; Graff et al., 2012; Roy et al., 2016). In addition, we have chronic visual GENUS mitigating these gene expression and protein phosphorylation defects throughout the CK-p25 and Tau P301S models, and immunohistochemical analysis using synaptic protein-specific markers (vGlut1, bassoon) in each control group. It was found to demonstrate synaptic density similar to that of mice. These gene expression changes and synaptic density rescue suggest that chronic GENUS transforms plasticity in the visual cortex and downstream brain regions, along with the enhanced low gamma interference we see in chronic visual GENUS.

The study of post-human AD samples, AD mice, iPSC models and primary cultured cells also implied intracellular transport, vesicle delivery, and destruction of endosomal function in AD (Millecamps and Julien, 2013; Small et al., 2017; Israel et al., 2012; Cataldo et al., 2000). The link between endocytosis and Aβ production has been well documented by many studies (Marks and McMahon, 1998; Cirrito et al., 2008; Schobel et al., 2008; Wu and Yao, 2009). Our unbiased transcriptome and proteomic data show that chronic GENUS refers to these changes in intracellular transport, vesicle delivery and endocytosis processes in gene expression and protein level post-translational modifications. Our results are consistent with a reduced initial endosomal reduction in CA1 of young 5XFAD mice following acute photogenetically driven gamma oscillation (Iaccarino et al., 2016). In addition, our data show that chronic GENUS reduced S774 dynamin 1 hyperphosphorylation in both neurodegenerative P301S and CK-p25 mouse models, which influenced the role of dynamin in endocytosis (Raimondi Et al., 2011; Armbruster et al., 2013).

Taken together, we believe that the structure (or "repair" of a wide range of genes and proteins that neuron survival enhanced by chronic GENUS is involved in modulating DNA damage response, autophagy, synaptic transmission, intracellular transport, vesicle transport and reduced neuroinflammation. "). Supports the extensive neuroprotective effects of chronic visual GENUS to promote neuronal survival and maintain neuronal function, improved new object recognition and spatial Morris underwater maze in CK-p25 mice and enhanced spatial underwater maze learning and memory in P301S mice and 5XFAD mice Including, finding improvement in behavioral performance in several mouse models after chronic GENUS. While we observed improved behavioral performance, chronic GENUS was observed in C57B1/6J, CK-p25 and P301S mice by body weight (FIGS. 11D-11F), by open field exploration as analyzed by marker corticosteroids. The anxiety or stress response was not altered (FIGS. 11A, 11C, 11G-11I). Interestingly, Zhang et al. (2015) demonstrated that chronic 2 Hz visual stimulation (6 hours per day for 4 weeks) improved cognitive performance, but did not alter Aβ1-42 in the target cortex of 3xTg mice.

Visual genus reduces neuroinflammation

The inflammatory process is known to play an important role in AD and neurodegeneration, and acute GENUS has been previously shown to induce remarkable morphological activity of microglia (Iaccarino et al., 2016). The specific role of microglia in neurodegeneration is complex and must be thoroughly investigated (because it can be beneficial or harmful), and recent studies have revealed that microglia phenotype can be altered as the disease progresses (Mathys et al., 2017). ; Lee et al., 2018; Ulland et al., 2017; Deczkowska et al., 2018). In the present disclosure, we performed a detailed morphological analysis using immunohistochemistry and microglia specific transcriptologic analysis in order to more closely investigate the basic processes affected by chronic GENUS. However, despite chronic GENUS altering microglia morphology, immune response, and catabolic processes across both CK-p25 and Tau P301S mouse models, it has been found that there are various types of phenotypes.

First, Iba1 immunostaining is highly proliferative (more microglia; more consistent with Mathys et al. (2017)) in CK-p25 mice and tends to clump together, which decreases after chronic GENUS. Excluding a subset of rod-like microglial cells in which total soma and primary spine volumes were higher in unstimulated CK-p25 mice, the total volume of microglial processes was reduced, and chronic GENUS reduced this abnormal microglia phenotype. Interestingly, rod-shaped microglia have recently been reported to be present in distributed brain damaged rat brain and human subjects with traumatic brain injury (Bachstetter et al., 2017; Taylor et al., 2014). Next, the microglia enters a phagocytosis state to absorb Aβ in 5XFAD mice, which together with morphological changes in microglia associated with reduced amyloid plaque number, several aspects of GENUS (visual, auditory or a combination of both). About (Iaccarino et al., 2016; Martorell and Paulson et al., accompanying submissions). In addition, the phagocytic status of microglia is accompanied by increased proteolysis (single catabolic process as identified by GO). The inflammatory response of microglia has recently been investigated in the context of neurodegeneration and AD. Our data suggest that chronic GENUS reduced the inflammatory response of microglia in CK-p25 mice and down-regulated genes such as those related to the MHC class II and adaptive immune systems known to be up-regulated in neurodegeneration. (Mathys et al., 2017). Perhaps as a result of this reduced inflammatory response, we have C1q (Hong et al., 2016), a marker of synaptic pruning, with an increase in the synaptic marker vGlut1 and bassoon showing the conservation of synaptic density with chronic GENUS. ). It is important to note that the accompanying Martorell and Paulson et al. demonstrate astrocyte responses and demonstrate astrocyte responses after chronic auditory GENUS and auditory and visual complex GENUS, and vasculature changes.

In summary, our data demonstrate that visual GENUS can propagate to multiple regions of the forebrain (SS1, CA1, PFC), eliciting responses from multiple cell types (neurons, microglia, astrocytes). Gamma tuning using visual stimulation represents a non-invasive strategy to provide protective effects against neurological disorders and improve cognitive function. We found that chronic visual GENUS reduced the loss of neurons and synapses and modified genes and proteins associated with synaptic function to support the behavioral improvements observed together. The present disclosure demonstrates the neuroprotective effect of chronic visual GENUS in AD mouse models affecting AD pathology in multiple brain regions and behaviors, demonstrating the overall efficacy of GENUS conferring neuroprotection.

Detailed description of the drawings

1A-1J show, in accordance with the disclosed invention, visual stimulation tunes gamma vibrations in multiple brain regions of a subject past the visual cortex.

In FIG. 1A, a 40 Hz visual stimulus with 50% duty cycle (12.5 ms on and 12.5 ms off) was delivered using an Arduino system. C57BL/6J mice that received a 40 Hz visual stimulation for an unstimulated or 1 hour period were then sacrificed and stained with the brain for c-Fos expression. For in vivo electrophysiology, microdrives were implanted in separate cohorts.

1B, c-Fos expression was quantified in a coronal brain slice 40 μm thick to evaluate neuronal activity. Representative c-Fos immunostaining image. Scale bar represents 50 μm. Right : The stimulation at 40 Hz time is visual cortex (V1; N = 4 mice/group. Independent sample t-test, T = -7.110, P =0.002), somatosensory cortex (SS1; T = Neuronal activity marker c-Fos was significantly increased in -5.239, P = 0.006), hippocampus (CA1; T = -4.989, P = 0.008) and prefrontal cortex (CC; T = -2.938, P = 0.01).

In Figure 1C, output spectra from C57BL/6J mouse LFP were recorded from V1, SS1, CA1 and PFC. The red line represents the recording during the visible 40 Hz visual stimulation presentation, while the blue line represents the blocked 40 Hz light flicker (LED array is covered to block light; see method; N = 7 mice).

In FIG. 1D, group gamma area output (40±5 Hz) was calculated from FIG. 1C. 40 Hz visual stimulation is V1 (Wilcoxon-Ranksum, Z = 5.9, P = 3.1 x 10 ^-9 ), SS1 (Z = 2.4, P = 0.018), CA1 (Z =) compared to the control condition (LED blinking is blocked). 3.4, P = 6.9 x 10 ^-4 ), and PFC (Z = 3.3, P = 9.2 x 10 ^-4 ).

In FIG. 1E, we implanted a custom tetrahedral tube microdrive into a C57BL/6J mouse, adjusted the quadrilateral tube to CA1 and isolated a single unit. The chart shows the spike probability over the 40 Hz phase and 40 Hz stimulation cycle in the blocked light.

In FIG. 1F, visual stimulation at 40 Hz compared to light blocking condition (Oc. LED) significantly increases the phase lock intensity of vertebral neuron spikes against local LFP as analyzed by mean result length (MRL). Appeared (N = 24 cells from 4 mice.Wilcoxon-Ranksum, Z = 2.5, P = 0.011).

In FIG. 1G, LFP interference between structures was quantified using a weighted phase delay index method (WPLI; N=7 mice), and recording sites were indicated.

In FIG. 1H, a group change of low gamma band (30-50 Hz) WPLI is shown with respect to FIG. 1G. 40 Hz visual stimulation significantly increased interference between V1-CA1 (Wilcoxon-Ranksum, Z = 2.2, P = 0.03), V1-SS1 (P = 0.021) and V1-PFC (Z = 2.5, P = 0.014) Induced.

1I is a schematic diagram of 80 Hz LED light transmission with 50% duty cycle (6.25 ms on and 6.25 ms off).

1J shows the output spectrum of the V1 LFP in C57Bl/6J mice receiving blocked light (Oc. LED) or 80 Hz visual stimulation, on the left. On the right, the output area centered at 80 Hz (±5 Hz) was not significantly different from the 80 Hz visual stimulus (N = 5 mice, Wilcoxon-Ranksum test, Z = 1.2, P = 0.22).

2A-2F show that, according to the disclosed invention, chronic 40 Hz (but not 80 Hz) visual blink stimulation reduces amyloid plaques beyond the subject's visual cortex.

Figure 2A shows amyloid plaque load in 5XFAD mice exposed to 40 Hz or 80 Hz visual stimulation for 1 hour per day for 7 days without irritation, as visualized by immunohistochemical staining of the D54D2 antibody. Representative images of V1, SS1 and CA1 in each condition, the scale bar shows 50 μm.

Figure 2b shows group quantification showing that 1 hour of GENUS per day for 7 days reduces amyloid plaques in V1, but amyloid plaques were not significantly altered in SS1 or hippocampal region CA1. 80 Hz visual stimulation exposure did not alter the level of amyloid plaques in V1 or CA1, whereas amyloid plaques in SS1 increased significantly. N=8 unstimulated, and 6 mice each in the 40 Hz and 80 Hz groups. Two-way ANOVA effect between groups F (2, 51) = 5.378, P = 0.0076. Bonferroni's post-multiple comparison test, *** P <0.001, * P <0.05.

2C shows a representative image of amyloid plaque load in 5XFAD mice exposed to the GENUS protocol unstimulated or extended to 22 days (1 hour per day). Scale bar represents 50 μm.

2D shows that GENUS on day 22 significantly reduced amyloid plaques in V1, SS1, CA1 and CC. N = 6 mice per condition. Two-way ANOVA effect between groups F (1, 40) = 51.00, P <0.0001. Bonferroni's multiple comparison test, *** P <0.001, ** P ≤ 0.01.

FIG. 2E shows a representative image of amyloid plaques in V1, SS1 and CA1 visualized from mice exposed to 80 Hz light flicker for 1 hour per day for no stimulation or 22 days, and the scale bar represents 50 μm.

2F is related to FIG. 2E and shows group data quantifying amyloid plaques in V1, SS1 and CA1. 6 mice per group N. No significant differences were found between groups in the bidirectional ANOVA measurements F (1, 30) = 0.0033, P = 0.9565.

3A-3J show that in accordance with the disclosed invention, chronic visual stimulation improves Alzheimer's disease related pathology and particularly reduces or prevents neurodegeneration in subjects.

FIG. 3A provides an experimental outline showing immunohistochemistry and phosphoprotein physicochemical analysis after GENUS for 22 days (1 hour per day) without irritation in P301S mice. WT cage litters that did not receive stimulation therapy were considered WT naive mice.

3B provides a representative image showing S202/T205 phosphorylated Tau immunostaining from the visual cortex. Scale bar 50 μm. Right : P301S Tau mice showed higher S202/T205 levels in V1, SS1, CA1 and CC, while GENUS on Day 22 significantly reduced S202/T205 in all areas investigated. N=7 WT naive mice, 8 unstimulated P301S mice and 7 GENUS stimulated P301S mice. Two-way ANOVA effect between groups F (2, 76) = 45.35, P <0.0001. Post multiple comparison test with Bonferroni calibration, **** P <0.0001, *** P <0.001, ** P <0.01, * P <0.05.

3C depicts a phosphoprotein physicochemical analysis of serine/threonine phosphorylated tau protein from the visual cortex. The heat map shows the S/T residue of the tau protein that is differentially phosphorylated in P301S compared to WT mice. Two-way ANOVA effect between groups F (2, 322) = 2146, P <0.0001. All phosphopeptides were mapped as described in the method. All S/T residues of Tau shown in the heat map were statistically significant between WT naive and unstimulated P301S. Statistical comparisons (P values) between unstimulated and GENUS stimulated P301S mice for specific residues are shown in the chart. GENUS reduced phosphorylation of Tau protein at 6 S/T sites and increased phosphorylation at S451. The commonly known human tau S/T site is shown on the top.

3D shows a representative image of the neuron marker NeuN in the visual cortex of P301S mice that received WT naive and unstimulated or GENUS. Scale bar represents 50 μm.

3E shows group data in which P301S mice show significant loss of neurons in V1, SS1, CA1 and CC compared to WT naive mice. The GENUS stimulated P301S group showed significantly reduced neurodegeneration. N = same as in FIG. 3B. Two-way ANOVA effect between groups F (2, 76) = 19.73, P <0.0001. Post multiple comparison test with Bonferroni calibration, *** P <0.001, ** P <0.01, * P <0.05.

3F provides an experimental overview showing induction of p25 expression for 42 days in CK-p25 mice. This was accompanied by GENUS for 1 hour per day in one experimental group, and the irritation control group was illuminated with room light. CK (CaMK2α-promoter x tTA) cage litters without stimulation therapy were considered as CK naive mice.

3G is a photomicrograph showing qualitative differences in brains between CK-naive mice, unstimulated mice and GENUS stimulated CK-p25 mice 42 days after induction. Right : CK-p25 mice showed reduced brain weight (ie, brain atrophy) compared to CK naive mice, whereas chronic GENUS partially relieved brain atrophy in CK-p25 mice. N=13 CK naive mice, 10 mice each for the unstimulated and GENUS CK-p25 groups. ANOVA F (2, 30) = 15.46, P <0.001. Post multiple comparison test with Bonferroni correction, **** P <0.0001, * P <0.05.

3H provides a representative image and size quantification of the lateral ventricle (outline), and the scale bar shows 1000 μm. Right : Compared to CK naive mice, CK-p25 mice showed abnormal ventricular dilatation, which was significantly reduced after chronic GENUS. N = 9 CK naives and 6 in each of the non-irritating and GENUS CK-p25 groups. ANOVA F (2, 18) = 12.36, P <0.001. Post multiple comparison test with Bonferroni correction, **** P <0.0001, * P <0.05. See also FIG. 9E.

3i provides representative images for neuron marker NeuN in the visual cortex of CK-p25 mice receiving WT naive and unstimulated or GENUS. Scale bar represents 50 μm.

3J shows that CK-p25 mice showed severe neuronal loss in V1, SS1, CA1 and CC, while chronic GENUS reduced neuron loss in CK-p25 mice. N = same as in FIG. 3G. Two-way ANOVA F (2, 72) = 31.38, P <0.0001. Post multiple comparison test with Bonferroni correction **** P <0.001, *** P <0.001, ** P <0.01, * P <0.05.

4A-4Q show that, according to the disclosed invention, chronic visual stimulation reduces inflammatory responses in the subject's microglia.

4A relates to CK-p25 mice that received GENUS for 1 hour every day for 42 days without irritation. Subsequently, the microcortex cells of the visual cortex were extracted and sequenced, followed by isolation using a sequence fluorescence-activated cell sorter (FACS; using CD11b and CD45 double positive standards), followed by RNA extraction and sequencing. Differently expressed genes (DEG) are shown in the volcanic plot. Group comparisons are shown at the bottom. Left : DEG between livers of CK naive and unstimulated CK-p25 mice, N = 4 animals per group. Right : DEG, N = 4 mouse groups between unstimulated and GENUS CK-p25 mice.

4B shows selected gene ontology (GO) terms for biological processes associated with the identified DEG. Upper : GO term associated with the upregulated (UP) gene in unstimulated CK-p25 mice compared to CK naive mice. Lower: GO terms associated with down-regulation in non-stimulated CK-p25 mice (DOWM) gene compared to CK naive mice.

4C shows selected GO terms related to DEG. Upper : GO term associated with the upregulated (UP) gene in GENUS CK-p25 mice compared to unstimulated CK-p25 mice. Lower: No irritation CK-p25 mice compared to those associated (DOWM) down-regulated in CK-p25 mice GO terms.

4D provides representative images showing the microglial markers Iba1 (green) and CD40 (red) immunostaining from the visual cortex. Scale bar 50 μm. N=7 CK naive mice, 6 mice each for the unstimulated and GENUS groups. Arrows and arrowheads indicate the branch volumes of rod-shaped dendritic and microglial processes, respectively.

4E shows that unstimulated CK-p25 mice showed a greater number of Iba1 positive cells than CK naive mice, and chronic GENUS significantly reduced Iba1 cell density in CK-p25 mice. ANOVA F (2, 16) = 17.79, P <0.0001.

In Fig. 4f, we performed a three-dimensional rendering of microglia using Imaris, and quantified the volume of soma and protrusion of microglia. N = 73 microglia per group from the same number of mice in Figure 4D. Frequency distribution of the volume (size) of small glioblastoma soma that did not show any statistical significance between groups (Kruskal-Wallis test, H = 0.3529, P = 0.8382).

FIG. 4G shows that the total volume of microglial processes (except for rod-like microglial cells) was significantly lower in stimulated CK-p25 mice compared to CK naive mice. GENUS stimulated CK-p25 mice showed no difference compared to CK naive mice (H = 9.224, P = 0.009).

4H shows quantification of the minimum distance between microglia. N=68 microglia from 9 CK naives, 131 microglia from 6 unstimulated mice and 95 microglia from 6 GENUS CK-p25 mice. The microglia of the unstimulated CK-p25 mice aggregated together compared to the CK naive mice, while GENUS significantly reduced microglia aggregation (H = 100.1, P <0.0001).

Figure 4i shows the quantification of the radial basal processes of rod-like microglia. N = 16 microglia from 9 CK naive, 19 non-stimulated and 19 microglia per group from 6 each of the GENUS CK-p25 groups. After GENUS of CK-p25 mice compared to unstimulated CK-p25 mice, the overall volume of rod-like microglial processes was significantly reduced. ANOVA F (2, 51) = 16.27, P <0.0001.

4J shows that the non-stimulated CK-p25 mice showed higher signal intensity of the interferon-reactive protein CD40 compared to the CK naive mice, whereas chronic GENUS significantly reduced the CD40 signal in the CK-p25 mice. ANOVA F (2, 16) = 36.84, P <0.0001.

4K provides representative images from the visual cortex showing green Iba1 and blue Hoechst. Scale bar 50 μm. The arrow head shows the complexity of the microglial processes.

Figure 4l shows that the P301S tau mouse showed a tendency to increase the total number of Iba1-positive cells compared to the WT mouse, but was not statistically significant. N=7 WT naive mice, 8 unstimulated P301S mice and 7 GENUS mice. ANOVA F(2,19) = 2.401, P = 0.1190.

Figure 4m is a frequency distribution chart showing the size of small glioblastoma cells. N=7 WT naive mice, 8 unstimulated P301S mice and 7 GENUS mice. A total of 58 microglia were analyzed per group. The total volume of microglial soma did not differ between groups. Kruskal Wallis test H = 0.04269, P = 0.9789.

4n, the volume of microglia protrusions was smaller in P301S tau mice compared to WT mice, while GENUS stimulated P301S mice showed protrusion lengths similar to those of WT mice. Kruskal Wallis test H = 7.895, P = 0.0193.

4O provides a representative image showing C1q (classical complement pathway that mimics the protein) immunostaining from the visual cortex. Scale bar 50 μm. Upper : N=7 CK naive mice, 6 mice each for the unstimulated and GENUS stimulated groups. Lower: N = 7 Marie naive WT mice, non-stimulated 8 P301S mice and 7 GENUS mouse.

Figure 4p shows that the stimulation CK-p25 mice showed higher C1q signal intensity compared to the CK naive mice, whereas the chronic GENUS stimulated CK-p25 mice significantly decreased the C1q intensity. ANOVA F (2, 16) = 13.39, P = 0.0004.

Figure 4q shows that the unstimulated P301S mouse showed higher C1q signal strength compared to the WT naive mouse, and that chronic GENUS did not differ between any groups. ANOVA F (2, 19) = 6.887, P = 0.005. Post multiple comparison test with Bonferroni correction at e, i, j, l, p, q **** P <0.0001, ** P <0.01,* P <0.05, n.s- not significant.

5A-5I, according to the disclosed invention, chronic visual stimulation alters synaptic function and intracellular transport in neurons.

In FIG. 5A, CK-p25 mice received GENUS for 1 hour per day for no stimulation or 42 days. The visual cortex NeuN positive (100,000) neuron nuclei were isolated using FACS and then RNA extracted and sequenced. DEG's heat map. The number of mice per group is indicated on the heat map. Upper left : DEG between CK naive and non-stimulated CK-p25 mice. Bottom left : The number of DEG and genes between unstimulated and GENUS stimulated CK-p25 mice is displayed on the right. Right : Chart showing overlapping of biological projections associated with down-regulated genes in unstimulated CK-p25 mice compared to CK naive mice, and the same biological projections associated with up-regulated genes after GENUS in CK-p25 mice.

In FIG. 5B, P301S mice were irritated or GENUS stimulated for 1 hour per day for 22 days. The visual cortex NeuN positive (100,000) neuron nuclei were isolated using FACS and then RNA extracted and sequenced. Heat map of differently expressed genes. The number of mice per group is indicated on the heat map. Upper left : DEG between WT naive and unstimulated P301S tau mice. Bottom left : The number of DEG and genes between unstimulated and GENUS stimulated P301S Tau mice is shown on the right. Right : Chart showing overlapping of biological protrusions associated with down-regulated genes in unstimulated P301S mice compared to CK naive mice, and identical biological protrusions associated with up-regulated genes after GENUS stimulation in P301S mice.

In Figure 5C, CK-p25 mice received stimulation or GENUS for 42 days. Total protein expression and S/T phosphorylation protein analysis was performed on visual cortical tissue using a TMT 10-plex kit and mass spectrometer. N = 3 CK naive mice, 3 unstimulated CK-p25 mice and 4 GENUS stimulated CK-p25 mice. Upper side : Venn diagram showing overlap of total RNA identified from neuron specific RNA seq (FIG. 5B, top) and total protein identified from LC-MS/MS. 92.73% of the identified proteins were found to be expressed in neurons. Bottom : Volcanic plot of proteins phosphorylated differently in CK-p25 mice compared to litter off ( left ) and GENUS ( right ) controls.

In FIG. 5D, P301S tau mice were irritated or GENUS stimulated for 22 days. Total protein expression and S/T phosphorylated protein analysis was performed on visual cortical tissue using a tandem mass tag (TMT) 10-plex kit (see method) and mass spectrometer (LC-MS/MS). N=3 WT naive, 3 unstimulated P301S mice and 4 GENUS stimulated P301S mice. Upper side : Venn diagram showing overlap of total RNA identified from neuron specific RNA seq (FIG. 5A, upper) and total protein identified from LC-MS/MS. 91.95% of the identified proteins were found to be expressed in neurons. Lower: littermates (left) and GENUS (right) volcano plot of the proteins differentially phosphorylated in tau P301S compared to the control mouse. Phosphorylated proteins with fold changes of ± 0.2 and adjusted P values of <0.05 were considered statistically significant.

5E shows GO terms for different S/T phosphorylated proteins in CK-p25 and P301S Tau mice after chronic GENUS.

FIG. 5F shows a representative image showing neurofilament heavy chain (NFH, neuron marker mainly expressed in axons) and Ser774 phosphorylated dynamin 1 immunostaining from the visual cortex. Neuronal processes were labeled using NFH. Scale bar 10 μm. N=7 CK naive mice, 6 mice each for the unstimulated and GENUS stimulated groups. Median : CK-p25 mice showed significantly higher levels of pS774-DNM1 signal strength compared to CK Naive, while GENUS significantly reduced this abnormal phosphorylation in CK-p25 mice. F (2, 16) = 38.551, P <0.0001. Bonferroni's multiple comparison test, ***P <0.001, **P <0.01. Right : Non-stimulated P301S Tau mice showed significantly higher levels of pS774-DNM1 signal strength compared to WT naive mice, whereas GENUS significantly reduced this abnormal phosphorylation in P301S mice. N=7 WT naive mice, 8 unstimulated P301S mice and 7 GENUS mice. ANOVA F (2, 19) = 18.69, P <0.0001. Post multiple comparison test with Bonferroni correction ** P <0.01, ns- not significant.

5G shows representative brain fragments immunostained for vesicular glutamic acid transporter 1 (vGlut1) from the visual cortex of CK naive, non-irritated and GENUS stimulated CK-p25 mice. Scale bar represents 10 μm.

Figure 5h shows that vGlut1 spot expression is significantly lower in unstimulated CK-p25 mice, whereas GENUS stimulated CK-p25 mice have higher levels of vGlut1 in V1, SS1, CA1 and CC compared to unstimulated CK-p25 mice. Indicates that the spots were shown. N=9 CK naive mice, and 6 mice each from the unstimulated and GENUS CK-p25 groups. Two-way ANOVA effect between groups F (2, 72) = 42.06, P <0.0001. Post multiple comparison test with Bonferroni correction **** P <0.0001, ** P <0.01,* P <0.05.

5i shows that the expression of synaptic spot vGlut1 was significantly lower in P301S compared to WT naive mice, and GENUS rescued vGlut1 expression in V1, CA1 and CC of P301S (rescued). N=7 WT naive mice, and 8 unstimulated P301S mice and 7 GENUS mice. Two-way ANOVA effect between groups F (2, 76) = 23.67, P <0.0001. Post multiple comparison test with Bonferroni correction **** P <0.0001, *** P <0.001, ** P <0.01,* P <0.05.

6A-6I show that, according to the disclosed invention, chronic visual stimulation alters behavior in a multi-object model of Alzheimer's disease.

In FIG. 6A, p25 expression was induced in two groups of CK-p25 for 42 days, only one of them received 1 hour daily GENUS (stimulation was performed between 9 am and 12 pm). Mice were subjected to open field (OF) and new object recognition (OR) tests on the 40th (OF) and 41st (OR) afternoons (3 pm to 7 pm). F and N refer to existing and new objects, respectively. Shows representative occupancy heat maps of OF and OR sessions. The color level maps to the range of the arena's location frequency (warm colors represent longer times and frequencies, while cold colors represent shorter times at specific locations).

6B shows that chronic visual GENUS did not affect the level of anxiety in CK-p25 mice. The time required at the center of the arena did not differ between CK naive mice, non-irritated and GENUS stimulated CK-p25 mice. ANOVA effect between groups F (2, 49) = 1.198, P = 0.3104.

6C shows that in CK naive and GENUS stimulated CK-p25 mice, the preference for new objects was significantly higher than in the existing objects, but the non-irritated CK-p25 mice did not show preference. Two-way ANOVA effect between old and new objects F (1, 70) = 7.742, P = 0.0069. T-test; CK Naive, T = 4.421, P = 0.0001; CK-p25 + no stimulation, T = 1.108, P = 0.0946; CK-p25 + GENUS, T = 3.306, P = 0.0017.

Figure 6D shows Morris water maze performance of p25 induced CK-p25 mice with or without GENUS for 42 days (MWM; between days 36 and 41 of p25 induction). Upper side : Chronic GENUS reduced the latency to find the platform during training in CK-p25 mice compared to unstimulated CK-p25 mice. Two-way ANOVA effect between groups F (2, 252) = 18.64, P <0.0001. Lower: in the probe test performed 24 hours after the final training session can be cross-platform, were more significant in GENUS stimuli received group compared to non-stimulated CK-p25 mice (left: inter-group effect F (2, 42) = 5.277 , P = 0.0090). The time spent in the target quadrant ( right ; intergroup effect F (2, 42) = 6.35, P = 0.0039) was significantly lower in the unstimulated CK-p25 mice compared to the CK naive mice.

FIG. 6E relates to P301S Tau mice that were irritated or exposed to GENUS for 1 hour per day for 22 days (9 am to 12 pm) and then evaluated in the OF (Day 20; 3 to 7 pm) and OR (Day 21) tests. . Shows representative occupancy heat maps of OF and OR sessions.

6F shows that GENUS did not affect the level of anxiety in P301S mice. The time spent at the center of OF was not different between the unstimulated, GENUS stimulated P301S and WT naive groups (ANOVA F (2, 36) = 1.189, P = 0.3163).

6G shows that WT, non-irritated and GENUS stimulated P301S mice all showed higher preference for new objects. Two-way ANOVA effect between old and new objects F (1, 80) = 88.61, P <0.0001. T-test; WT naive, T=6.525, P<0.00001; P301S + no stimulation, T = 2.602, P = 0.0054; P301S + GENUS, T = 10.65, P <0.00001.

Figure 6h shows the performance of MWM performance of P301S Tau mice stimulated with WT naive, unstimulated or GENUS. Upper side : Chronic GENUS reduced the latency to find the platform during training in P301S mice compared to unstimulated P301S mice. Two-way ANOVA effect between groups F (2, 225) = 3.782, P = 0.0242. Lower: The cross-platform in a probe test (left: F (2, 45) = 2.872, P = 0.067) , and time in the target quadrant in the probe test (on the right: F (2, 45) = 3.115, P = 0.054).

In Figure 6i, 5XFAD mice with or without GENUS (1 hour daily for 22 days) were tested for MWM performance. Left : Waiting time to find the platform while training. Two-way ANOVA effect between groups F (2, 273) = 16.97, P <0.0001. Number of platform crossings in the probe test ( center : effect between groups F (2, 39) = 4.702, P = 0.0148) and time spent in the target quadrant ( right : effect between groups F (2, 39) = 7.289, P = 0.0020) Was significantly lower in unstimulated CK-p25 mice.

7A-7I show that, according to the disclosed invention, chronic visual stimulation tunes gamma vibrations beyond the visual cortex in a neurodegenerative mouse model.

FIG. 7A shows electrolyte lesions to identify recording sites in mice used for post-recording of primary FIGS. 1C and 1D. Representative images show recording sites from V1, SS1, CA1 and PFC.

In FIG. 7B, CDK5 activator p25 was induced in CK-p25 mice for 6 weeks, followed by microdrive transplantation and LFP recording.

In FIG. 7C, zone output (35-45 Hz) was significantly lower in CK-p25 mice compared to WT mice of the same age in V1, CA1 and PFC (all regions P <0.01). 40 Hz visual stimulation showed 40 Hz output from V1 (Wilcoxon-Rank sum; P = 0.0022), CA1 (P = 0.001) and PFC (P = 0.002) during 40 Hz light blink exposure in CK-p25 mice induced for 6 weeks. Increased.

In FIG. 7D, microdrives were implanted in 8 month old P301S Tau mice.

7E shows that a significant increase in power (35-45 Hz) was observed during 40 Hz light stimulation in V1 (P = 6.01E-05) and PFC (P = 4.11E-05).

FIG. 7F relates to C57BL6/J that was not stimulated or received GENUS for 1 hour per day for 42 days. Right : During 40Hz visual stimulation, V1 (Wilcoxon-Ranksum test; P = 1.54E-06), SS1 (P = 2.88E-04), CA1 (P = 0.04) and PFC (P = 3.39E-06) 35 A significant increase in ˜45 Hz zone output was observed.

In FIG. 7G, C57BL6/J mice were not stimulated or received GENUS 1 hour/day for 42 days. Mice were visually stimulated at 40 Hz on day 43 and LFP was collected. During GENUS, significant increases between zones ranged from 30 to 50 Hz between V1 to CA1 (P = 0.002), V1 to SS1 (P = 0.008), CA1 to PFC (P = 0.04), and V1 to PFC (P = 0.002). Low gamma interference (measured with a weighted phase delay index) was observed. There was no difference in low gamma WPLI between CA1 and SS1 (P = 0.18).

In FIG. 7H, p25 was induced in CK-p25 mice for 6 weeks. CK-p25 mice induced at 6 weeks experienced stimulation (from day 43), irritation or 1 hour/day for 42 days. Right : Significant increase in low gamma zone output (35-45 Hz) was studied in CK-p25 mice [V1, P = 0.0017), CA1 (P= 0.0379), PFC (P= 0.0030)] and all brain zones Was observed (measured on day 85).

7i shows that a significant increase in low gamma interference was also evident between V1 to CA1 (P <0.01), CA1 to PFC (P <0.01), and V1 to PFC (P <0.01).

8A-8I show that, according to the disclosed invention, chronic visual stimulation reduces AD related pathology in 5XFAD mice beyond the visual cortex.

8A is a schematic diagram showing a visual blink stimulation setting. An array of light emitting diodes (LEDs) was present on the open side of the cage and was driven to flash at a frequency of 40 Hz with a square wave current pattern using an Arduino system. Since the mice move freely within the cage (total floor area 83 in²), the light intensity they receive ranges from about 1500 to 300 lux.

In FIG. 8B, 10-month-old 5XFAD mice received GENUS for 1 hour per day for no irritation or 22 days. Representative images show amyloid plaques red and nuclear stained Hoechst blue from slices containing the hippocampus and somatosensory cortex. Scale bar 1000 μm. Related to the main diagrams 2c to 2d.

In FIG. 8C, 10-month-old 5XFAD mice received GENUS for 1 hour per day for 22 days without irritation. Representative images show amyloid plaques red, neuron marker NeuN green and nuclear stained Hoechst blue from the target cortex. Scale bar 50 μm. Related to the main diagrams 2c to 2d.

8D shows that the number of plaques in GENUS stimulated 5XFAD mice was significantly lower in V1, SS1, CA1 and CC than in unstimulated 5XFAD mice. Related to the main diagrams 2c to 2d. N = 6 mice per condition. Two-way ANOVA effect between groups F (1, 40) = 30.01, P <0.0001. *** P <0.001, ** P <0.01, * P <0.05.

In FIG. 8E, 9 month old unstimulated 5XFAD mice compared to the same age WT litter offspring showed a significant decrease in neuronal density in CA1 and CC. GENUS significantly reduced neuronal loss in 5XFAD mice in CC. N = 9 WT naive, 6 5XFAD + irritation, and 6 5XFAD + GENUS mice. Two-way ANOVA effect between groups F (2, 72) = 14.93, P <0.0001. Bonferroni multiple comparison test, ***P <0.001, **P <0.01, *P <0.05.

In FIG. 8F, 10-month-old 5XFAD mice received GENUS for 1 hour per day for 22 days without irritation. A representative image shows the synaptic marker bassoon. Scale bar 10 μm.

In Figure 8G, 5XFAD mice showed a significant reduction in synaptic marker bassoon staining at CA1 and CC, which was significantly reduced by 40 Hz GENUS. N = 9 WT naive, 6 5XFAD + irritation, and 6 5XFAD + GENUS mice. Two-way ANOVA effect between groups F (2, 72) = 16.18, P <0.0001. Bonferroni multiple comparison test, ***P <0.001, **P <0.01, *P <0.05.

8H provides a full length immune blot showing the expression levels of the full length APP protein and endogenous GAPDH control. N=5 WT naive mice, 3 unstimulated 5XFAD mice and 4 GENUS stimulated 5XFAD mice.

8i shows that APP protein expression is significantly higher in 5XFAD mice compared to the WT naive control. There was no difference between unstimulated and GENUS stimulated 5XFAD mice. Note that we did not measure the C/N fragment of the APP protein. C-terminal specific APP antibody, one way ANOVA F (2, 9) = 4.436, P = 0.046. N-terminal specific APP antibody, one way ANOVA F (2, 9) = 13.194, P = 0.002. Bonferroni multiple comparison test, **P <0.01.

9A-9G show that according to the disclosed invention, chronic visual stimulation improves AD related pathology in P301S and CK-p25 mice.

9A provides a full length immunoblot showing the expression levels of total tau protein and GAPDH from the visual cortex of unstimulated, GENUS stimulated P301S Tau mice, and WT naive litters of the same age. Right : Total tau expression was increased in P301S tau mice compared to WT naive mice; Tau levels between unstimulated and GENUS stimulated P301S mice were not different. N = 3 WT naive, 3 irritated and 4 GENUS stimulated P301S mice. ANOVA F (2, 7) = 173.275, P <0.0001. Bonferroni multiple comparison test, ***P <0.001.

9B shows that there was no difference in brain weight between WT naive, unstimulated P301S and GENUS stimulated P301S mice. There was a tendency for abnormal swelling of the lateral ventricles in P301S tau mice compared to WT naive mice. ANOVA F(2,19) = 2.761, P = 0.079.

9C provides a full length immune blot showing the expression levels of p25 +GFP (fusion protein), p25, p35 and GAPDH from the visual cortex of CK naive, unstimulated and GENUS stimulated CK-p25 mice. Right : There is a significant increase in p25+ GFP protein in CK-p25 mice compared to CK mice of the same age (one way ANOVA, F (2,12) = 13.065, P = 0.002), but unstimulated and GENUS stimulated CK- p25 did not differ in p25+ GFP expression. Similarly, p25 expression levels were significantly higher in CK-p25 mice compared to CK naive mice, but there was no difference between unstimulated and GENUS stimulated CK-p25 mice (one way ANOVA, F (2, 12) = 3.581, P = 0.025). Bonferroni multiple comparison test, ***P <0.001, **P <0.01, *P <0.05.

9D provides representative images showing the thickness of the visual cortex in CK naive, non-irritating CK-p25 and GENUS stimulated CK-p25 mice. Nuclear staining Hoechst is marked in blue and red in the neuron marker NeuN. Right : The bar chart shows the difference in visual cortical thickness between groups. Two-way ANOVA effect between groups F (2, 36) = 12.93, P <0.0001. Bonferroni multiple comparison test, ***P <0.001, **P <0.01, *P <0.05.

9E provides a representative image showing qualitative differences between CK naive, unstimulated and GNEUS stimulated CK-p25 mice showing changes in hippocampal volume, cortical thickness and ventricular size. Related major degrees 3f to 3h.

9F shows that, across all brain regions tested, GENUS did not alter p25 +GFP fusion protein expression levels (number of p25: GFP expressing neurons) compared to unstimulated CK-p25 mice, independent sample t-test between groups, P>0.05.

9G provides a representative image showing the expression of the DNA double-strand break marker γH2Ax from CA1 in CK naive, unstimulated and GENUS stimulated CK-p25 mice. The γH2Ax positive nuclei were not clear in CK naive mice, and thus were compared between unstimulated and GENUS stimulated CK-p25 mice. N = 6 mice per condition. Scale bar 100 μm. Right : GENUS is V1 (independent sample t-test; T = 4.418, P = 0.0006), SS1 (T = 2.944, P = 0.013), CA1 (T = 2.664, P = 0.0143) and CC (T = 1.883, P = 0.055), the γH2Ax positive nuclei were significantly reduced.

10A-10N show that according to the disclosed invention, chronic visual stimulation transforms microglia and enhances intracellular transport and synaptic transmission in neurons.

10A shows microglia separation profiles in FACS from representative CK naive, unstimulated and GENUS stimulated CK-p25 mice. Related Principles 4a, 4c.

10B shows microglial separation profiles in FACS from representative WT naive, unstimulated and GENUS stimulated P301S Tau mice.

In FIG. 10C, differently expressed genes (DEG) are shown in a volcanic plot. Group comparisons are shown on the right. Upper : DEG between WT naive and unstimulated P301S tau mice, N = 5 mice per group. Middle top : DEG, N=5 mouse group between unstimulated and GENUS stimulated P301S mice.

10D shows the top 7 processed gene ontology (GO) terms for the biological processes associated with the identified DEG. Group comparisons are shown from the top.

10E provides representative images showing green Iba1 and red CD40. The merged images presented in principal figure 4d were separated for clarity.

10F shows the neuronuclear separation profile in FACS from representative CK naive, unstimulated and GENUS stimulated CK-p25 mice. Group mean and SEM of% NeuN are compared to total nuclei: CK Naive, 50±1.48; Unstimulated CK-p25, 40.98 ± 0.719; GENUS stimulated CK-p25 mice, 45.08 ±1.691. ANOVA F (2, 12) = 11.55, P = 0.0016. Bonferroni post-test, CK naive vs. non-irritating CK-p25, P = 0.001' CK naive vs. GENUS stimulated CK-p25 P = 0.0609. GENUS reduced the loss of neuronuclei in CK-p25 mice.

10G shows neuronuclear separation profiles in FACS from representative WT naive, unstimulated P301S and GENUS stimulated P301S mice. The group mean of the percentage of NeuN positive nuclei found and the standard error of the mean (SEM) are compared to the total Hoechst positive nuclei: WT Naive, 52.45±2.03; Unstimulated P301S, 45.17 ± 1.18; GENUS stimulated P301S, 50.38 ± 2.09. ANOVA F (2,18) = 4.97, P = 0.0191. Bonferroni post-test, WT naive vs. unstimulated P301S, P = 0.028; WT irritation vs. GENUS stimulated P301S mice, P = 0.99. GENUS reduced the loss of neuronuclei in P301S Tau mice.

10H provides a bar graph showing the percentage of Neuron positive nuclei compared to the entire nucleus as in FIGS. 10F and 10G. Upper side : Bar graph comparing WT naive, unstimulated and GENUS stimulated P301S mice. N = 7 mice per group. ANOVA F (2, 18) = 4.971, P = 0.0191. Bottom : Bar graph comparing CK naive, unstimulated and GENUS stimulated CK-p25 mice. N = 5 mice for each group. ANOVA F (2, 12) = 7.72, P = 0.0070.

In FIG. 10I, genes up-regulated in P301S tau mice and CK-p25 mice were selected after chronic GENUS based on neurotransmitter transport and vesicle-mediated transport clusters with 60 genes. Note that these genes were upregulated only in CK-p25, P301S or in both mice after GENUS compared to unstimulated mice (analyzed by metascape). A protein-protein interaction network map (analyzed by Strings after Cytoscape V3.6.1; enriched P value, 6.66E-15) is shown in tightly linked additional functional enriched GO terminology.

In FIG. 10J, the visual cortex from unstimulated P301S tau-tg mice and GENUS-stimulated mice for 1 hour/day for 22 days was analyzed by S/T phosphoprotein physics. The heat map shows differently phosphorylated proteins between unstimulated and GENUS stimulated P301S mice involved in synaptic transmission and intracellular transport.

In FIG. 10K, the heat map shows different phosphorylated proteins involved in synaptic transmission (chemical synaptic transmission, trans-synaptic signal transmission) between unstimulated and GENUS stimulated CK-p25 mice. It indicates the name of the gene (the protein of interest) and the specific phosphorylation-site.

In Figure 10L, P301S tau mice were irritated or GENUS stimulated for 22 days. Total protein expression and S/T phosphorylated protein analysis was performed on visual cortical tissue using a tandem mass tag (TMT) 10-plex kit (see method) and mass spectrometer (LC-MS/MS). N=3 WT naive, 3 unstimulated P301S mice and 4 GENUS stimulated P301S mice. Phosphorylated proteins with fold changes of ± 0.2 and adjusted P values of <0.05 were considered statistically significant. GO terms of biological processes associated with different S/T phosphorylated proteins in CK-p25 and P301S Tau mice were compared to each control mouse.

10M- Upper : Western blot shows pS774 DNM-1, DNM-1, DNM-3 and GAPDH from CK naive, unstimulated and 40 Hz GENUS stimulated CK-p25 mice. pS774DNM-1 levels were significantly higher in CK-p25 mice compared to CK naive mice, while 40 Hz GENUS significantly reduced pS774DNM-1 in CK-p25 mice. N = 4 CK naive mice, 5 unstimulated CK-p25 mice and 4 40 Hz GENUS stimulated CK-p25 mice (ANOVA F (2,12) = 5.836, P = 0.021). Bottom : The bar chart shows group differences. Note that this is an independent confirmation. Related Principles 5f.

10N - Upper : Western blot shows pS774 DNM-1, total DNM-1, DNM-3 and GAPDH from WT naive, unstimulated and 40 Hz GENUS stimulated P301S mice. Note that this is an independent confirmation. *P <0.05. Related Principles 5f.

11A-11J show behavioral properties of effects of acute and chronic visual stimuli on a subject, in accordance with the disclosed invention.

11A top : shows heatmap of C57BL6/J mouse occupancy in 10 min bin with or without GENUS. There was no clear difference in exploratory behavior during the first 10 minutes of pre-stimulation baseline. N = 6 mice per group. T-test, T = 0.3173, P = 0.7576. Similarly, the systematic change in velocity over the 40 Hz stimulation period was not detectable when compared to the unstimulated group. Bottom : Plots show speed every minute for 30 minutes. Two-way repeated measures ANOVA: exploration over 30 minutes, F (29, 290) = 6.747, P <0.0001; Interaction between unstimulated and GENUS stimulated WT mice, F (29, 290) = 0.9354, P = 0.5652.

In FIG. 11B, C57BL6/J mice (4 months old) were irritated or irradiated with 40 Hz light for 1 hour and immediately injected with picrotoxin and placed in OF. Left : The distance traveled after the injection of picrotoxin showed no difference between the GENUS and non-irritated groups. N = 4 mice per group. Repeated measurements ANOVA F = 0.527, P = 0.717. Right : Lacine score. 0 points, normal behavior; 1 point, immobility and stiffness; 2 points, head bobbing; 3 points, intermittent spasm and support of the forelimbs; 4 points, continuous support and fall; 5 points, seizures of the ligament spasm; 6 points, death. The difference in seizure sensitivity between the unstimulated and GENUS groups was unclear. Repeated measurements ANOVA F = 0.429, P = 0.994.

In Figure 11C, 3 days after habituation of the new object, WT mice were tested for NOR. Left : Time spent exploring existing and new objects every minute for a 30-minute period. New object identification rates overlapped. Middle : The histogram shows the cumulative (30 minutes total) exploration of existing and new objects. Both unstimulated and GENUS-stimulated mice spent significantly longer time exploring new objects than existing ones. There was no difference between the groups, suggesting that acute 40 Hz light flicker did not affect the mouse's ability to identify new objects. N = 6 mice per group. New object preference accumulated over existing objects: no irritation, T (6) = -13.055, P = 0.00004; GENUS T (5) = -20.818, P = 0.000004. New object preference between groups, t-test T =-0.314, P = 0.760. Right : Histogram of total distance traveled, showing that GENUS did not alter motility behavior in WT mice. Total travel distance, t-test T = 0.5096, P = 0.6214.

In FIG. 11D, C57BL6/J mice received GENUS for 1 hour per day for 7 days without irritation. Mice were weighed 7 days and 1 hour prior to the daily stimulation paradigm for 1 day after stimulation therapy. N=14 unstimulated mice and 12 GENUS stimulated mice. Bar charts show body weight for 7 days. There was an overall increase in body weight over several days in both groups (F (6,144) = 2.889, P = 0.011), but there was no difference in body weight between the unstimulated and GENUS stimulated groups (F (6, 144) = 1.327, P = 0.249).

11E showed significant differences in weight between WT naive and P301S tau mice (two-way ANOVA effect F (2, 72) = 8.947, P = 0.0003 between groups), but chronic GENUS (22) compared to unstimulated P301S tau mice 1 hour/day during the day) did not affect the weight of P301S. N=13 WT naive, 14 irritated and 12 GENUS stimulated P301S mice.

Figure 11f shows that chronic GENUS did not affect the body weight of CK-p25 mice compared to unstimulated CK-p25 mice (inter-group two-way ANOVA effect F (2, 122) = 2.487, P=0.0874). N=25 CK naive mice, 21 unstimulated and 18 GENUS stimulated CK-p25 mice.

Figure 11G relates to the main figure 6F. N = same as in FIG. 6F. In the open field test, the total travel distance was significant between groups. ANOVA F (2, 36) = 10.27, P = 0.0003. However, there was no difference in GENUS stimulated P301S mice compared to unstimulated P301S (P> 0.99).

Figure 11h relates to the main figure 6b. N = same as in FIG. 6B. The open field test shows the total distance traveled. There was no difference in total travel distance during the open field in stimulated and GENUS stimulated CK-p25 mice. ANOVA F (2, 49) = 0.01552, P = 0.9846.

FIG. 11 i- Left : Plasma corticosteroid levels were not different between unstimulated and 7 day GENUS stimulated WT mice. N = 12 mice per group. T = 1.17, P = 0.255. Middle : While p25 was induced in CK-p25 mice, the mice received GENUS for 1 hour daily for unstimulated or 42 days. The number of mice per group is shown on the chart. Plasma corticosteroid levels were not different between CK naive, unstimulated and GENUS stimulated CK-p25 mice (F (2, 35) = 0.4871, P = 0.6185). Right : Plasma corticosterone in P301S Tau mice (22 days stimulation) was also comparable between WT naive, unstimulated P301S and 22 days GENUS stimulated P301S mice (F (2, 35) = 0.6874, P = 0.5096). . The number of mice in each group is shown accordingly.

11J relates to principal FIG. 7. Average speed at which the mouse swam in the Morris underwater maze test from all experiments. Left : There was no difference between any groups over the 6 day training period in CK-p25 mice (FIG. 7D ). Two-way ANOVA effect between groups F (2, 252) = 0.3832, P = 0.6821. Middle : There was no difference between any groups over the 5 day training period in P301S tau mice (FIG. 7H ). Two-way ANOVA effect between groups F (2, 225) = 0.5726, P = 0.5651. Right : The swimming speed was significantly different in the 5XFAD mouse cohort (FIG. 7i ). Two-way ANOVA effect between groups F (2, 273) = 24.24, P<0.0001. Multiple comparisons with Bonferroni correction. There were no differences on Days 1-4. Day 5: WT Naive vs. 5XFAD + Irritation, P >0.9999. WT Naive vs 5XFAD + 40 Hz GENUS, P = 0.0022. 5XFAD + no stimulation vs 5XFAD + 40 Hz GENUS, P = 0.0483. Day 6: WT Naive vs. 5XFAD + No Stimulation, P = 0.0005. WT Naive vs. 5XFAD + 40 Hz GENUS, P = 0.0037. 5XFAD + Stimulus vs 5XFAD + 40 Hz GENUS, P> 0.9999. Day 7: WT Naive vs. 5XFAD + Stimulation, P <0.0001. WT Naive vs. 5XFAD + 40 Hz GENUS, P = 0.0043. 5XFAD + no stimulation vs 5XFAD + 40 Hz GENUS, P = 0.5716.

12A-12C show that according to the disclosed invention, chronic visual stimulation at 80 Hz did not affect the Morris underwater maze in 5XFAD mice.

12A shows the performance in MWM of 5XFAD mice exposed to 80 Hz stimulation for 1 hour per day for 22 days. The number of mice per group is indicated in the chart legend. The latency to find the platform during training did not differ between the irritated and 80 Hz stimulated 5XFAD groups (Two-way ANOVA F (2, 180) = 13.33, P <0.0001), but these two groups were WT naive mice Compared to this, it took more time to find the platform.

12B shows the number of platform crossings during probe inspection. F (2, 30) = 3.622, P = 0.0390. Multiple comparisons with Bonferroni correction. *P <0.05.

12C shows that the time spent in the target quadrant during the probe test was significantly less in the irritated and 80 Hz stimulated 5XFAD mice compared to the WT naive mice. F (2, 30) = 5.643, P = 0.0083. Compared to WT naive mice, multiple comparisons with Bonferroni correction, stimulated-P = 0.014, 80 Hz-P = 0.0371. *P <0.05.

Experiment and analysis details

STAR METHODS

Animal model

All experiments were approved by the Animal Care Committee of the Department of Comparative Medicine at the Massachusetts Institute of Technology. C57BL6, Tg (Camk2a-tTA), Tg (APPSwFlLon, PSEN1*M146L*L286V), Tg (Prnp-MAPT*P301S) PS19, and Fos-tm1.1 (cre/ERT2) were obtained from Jackson Laboratories. Tg (tetO-CDK5R1/GFP) was produced in our laboratory. All transgenic mice were bred and maintained in our animal facility.

The C57BL/6J mice used were 3, 10 or 17 months of age. CK-control and CK-p25 (Camk2a-tTA bred with tetO-CDK5R1/GFP) mice were raised with doxycycline-containing food. Normal rodent food was provided to induce p25-GFP transgene expression. Typically, p25 expression was induced for 6 weeks. At the start of the experiment, P301S Tau mice were 7 months old and 5XFAD mice were 9 or 11 months old. All mice were housed in groups (2-5 mice per cage) except microdrives were implanted. All experiments were performed using litters of the same age. Immunostaining and electrophysiological experiments were performed twice using the total number of mice as defined in the figure legend. All behavioral experiments described herein (except Morris Morris maze in high and low WT mice and Tau P301S mice) were performed once, using the total number of animals in the animal as defined in the legend. Instead of using a statistical method to pre-determine sample size, we use group sizes from similar studies previously published in laboratory settings (Iaccarino et al., 2016; Nott et al., 2016) to keep the intergroup deviations to a minimum. I chose as much as possible. The box plot in FIG. 1 shows the middle and upper (75%) and lower (25%) quartile ranges. Unless stated, all plots in FIGS. 2-6 with error bars are reported as mean±SEM and all samples are reported as the number of mice (N).

40 Hz light flicker stimulation

Light blink stimulation was delivered as previously described (Iaccarino et al., 2016). Mice were transported from the storage room to the blink treatment room located on the adjacent floor of the same building. After habituation under dim lighting for 1 hour before starting the experiment, mice were introduced into the test cage (similar to the home cage except for no bedding and covering the three sides with a black sheet). The mouse was able to move freely in the cage, but was unable to access food or water during the light flashing for 1 hour. An array of light emitting diodes (LEDs) was present on the open side of the cage and was driven to flash at a frequency of 40 Hz with a square wave current pattern using an Arduino system. After 40 Hz light blink exposure for 1 hour, the mice were returned to their home cage and allowed to rest for an additional 30 minutes before being transported back to the maintenance room. Normal room lighting control mice were similarly exposed to similar cages with food and water restrictions, but they experienced only normal room lighting.

Organization preparation

Immunohistochemistry: Mice were myocardial infarction with 40 ml of ice-cold phosphate buffered saline (PBS) and 40 ml of 4% paraformaldehyde was added to PBS. Bromine was removed and fixed overnight with 4% PFA at 4° C. and transferred to PBS prior to sectioning.

Western blotting: The visual cortex was dissected, rapidly frozen in liquid nitrogen and stored in a -80°C freezer until treatment. Samples were homogenized using a glass homogenizer with RIPA (50 mM Tris HCl pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% sodium dioxycholate, 0.1% SDS) buffer. The protein concentration of the sample was quantified using Bio-Rad protein assay. Proteins of the same concentration were prepared and SDS-sample buffer was added.

Immune tissue chemistry

The brain was mounted on a vibratom stage (Leica VT1000S) using the prepared super-strong adhesive and 40 μm section. The pieces were then washed with PBS and blocked at room temperature for 2 hours using 5% normal donkey serum prepared in PBS containing 0.3% Triton-X100 (PBST). The blocking buffer was aspirated and the pieces were incubated with a suitable primary antibody (prepared in fresh blocking buffer) overnight at 4° C. in a stirrer. Then, washed three times with blocking buffer (10 minutes each), followed by incubation with Alexa Fluor 488, 555, 594 or 647 conjugated secondary antibody for 2 hours at room temperature. After washing three times (15 minutes each) with blocking buffer and once (10 minutes) with PBS, the pieces were mounted with fluoromount-G (electron microscopy science).

The following secondary antibody combinations were used: (1) Alexa Fluor 488, 594 and 647, (2) Alexa Fluor 555 and 647, and (3) Alexa Fluor 594 and 647.

Imaging and quantification

Images were acquired using an LSM 710 or LSM 880 confocal microscope (Zeiss) with 5, 10, 20, 40 or 63 times objectives at the same settings for all conditions. Images were quantified using ImageJ 1.42q or Imarisx64 8.1.2 (Bitplane, Zurich, Switzerland). For each experimental condition, two coronal sections from the indicated number of animals were used. Average values from two images per mouse were further used for quantification. Quantification was performed by blinded experimenters for genotyping and treatment conditions wherever possible.

NeuN, c-Fos and GFP positive cell counting: All images were acquired by Z-stack and quantified at 10 per image. The sum of all two means and all counts was calculated using ImageJ. NeuN counting from P301S Tau-tg and 5XFAD was performed by an experimenter blinded to treatment conditions.

γH2Ax positive cell counting: Cells were counted using ImageJ's multi-point tool.

vGlut1, Bassoon Spot: LSM 880 with 63x objective lens and 3x additional zoom acquired images. Both the visual and somatosensory cortex, the deep layer from the CA1 radiation layer (mainly 4 and 5) and the layer 5 of the ventral cortex were targeted. Single plane images were acquired. The number of vGlut1 spots was quantified using ImageJ's particle count plug-in.

pS202/T205 Tau Intensity: Obtained using LSM 710 (40 images from each field) with a 40-fold objective lens z-stack with a total slice thickness of 40 μm. All images were compressed/reduced and signal strength was measured in ImageJ.

pS774-DNM1 intensity: LSM 880 with 63x objective lens and additional 3x zoom was used to obtain a z-stack with a total slice thickness of 40 μm (40 images from each field). All images were compressed/reduced and the signal strength of the spots was measured in ImageJ.

Plaques: The D54D2 antibody stained the total plaque intensity and the number of plaques was quantified by the experimenter blinded to genotype and treatment conditions. The entire 40 μm piece was a z-stack imaged at 0.5 μm intervals, merged and signal strength was measured using ImageJ. Plaques were counted using ImageJ's particle analysis tool with a threshold of 5 μm ² . All plaque intensity and plaque counting was performed by an experimenter blinded to treatment conditions.

C1q intensity: obtained using LSM 710 (40 images from each field) with a 40-fold objective lens z-stack with a total slice thickness of 40 μm. All images were compressed/reduced and signal strength was measured in ImageJ.

CD40: LSM 880 using a 63x objective lens was used to obtain a z-stack with a total slice thickness of 40 μm (40 images from each field). All images were compressed/reduced and signal strength was measured in ImageJ.

Lateral ventricle: Perfect coronal fragments were imaged at -2.0 bregma (beak to tail) using an LSM 710 microscope with a 5x objective lens. ImageJ's free-form selection tool was used to draw a contour covering the entire area of the lateral ventricle and measure the area of the LV.

Microglia: Iba1 immunoreactive cells were considered microglia. It was acquired using an LSM 710 (40 images from each field) with a 40-fold objective lens z-stack with a total slice thickness of 40 μm. Imaris were used for 3D rendering of the images to quantify the total volume of soma and dendritic microglia. Iba1 aggregation analysis was performed in an ImageJ 3D rendering plugin. The minimum distance between Iba1 was calculated for every microglia in the image. All images were compressed/reduced and the total number of Iba1-positive cells was quantified using ImageJ.

Cortical thickness: Perfect cortical columns were imaged using an LSM 710 microscope with a 5x objective lens. The distance between the outer cortical border of the cerebral cortex and the cortical side was measured using ImageJ.

Brain weight measurement: Mice were cardiovascular perfused with PBS followed by 4% PFA and brains were fixed postmortem with 4% PFA overnight. Brains were washed with PBS and excess was removed prior to weighing on a wet laboratory high precision scale (Mettler Toledo, accurate to 1 mg). Another cohort of mice was sacrificed, the brain was rapidly frozen in liquid nitrogen and brain weight was measured. Brain weights were normalized to the CK naive brain in these two independent measurements.

Western blotting

6-8 μg of protein was loaded onto a 6, 8, 10 or 15% polyacrylamide gel and electrophoresed. The protein was transferred from acrylamide gel to nitrocellulose membrane (Bio-Rad) at 100 V for 120 minutes. The membrane was blocked with milk (5% w/v) diluted in PBS containing 0.1% Tween-20 (PBSTw), and then cultured overnight at 4°C with primary antibody. The next day, washed three times with PBSTw and incubated with secondary antibody (GE Healthcare) linked to horseradish peroxidase for 60 minutes at room temperature. After washing three more times with PBSTw, the membrane was treated with a chemiluminescent substrate and the film developed. Signal intensity was quantified using ImageJ 1.46q and normalized to the value of a control protein such as β-actin, GAPDH or total protein corresponding to phosphorylated protein analysis.

Microdrive implantation and in vivo electrophysiology

Tungsten wire electrode drive: Microdrive is a 3D printed drive base with perfluoroalkoxy coated tungsten wire electrode (50 μm bare diameter, 101.6 coated diameter, AM Systems) and Neuralynx electrode interface board (EIB-36) It was custom-made using. Polyimide tubes were used to protect the electrodes and reduce electrical noise. The electrodes were visual cortex (adjusted to the bregma, anterior-posterior (AP), -3.0; medial-external (ML), +2.0), SS1 (AP, -2.0; ML, +2.3), CA1 zone of the hippocampus ( AP, -1.8; ML, +1.5) and layers 3 or 4 of the target region of the frontal cortex (AP, +1.0; ML, +0.2). The reference electrode was placed in the cerebellum.

Quadrupole drive: The custom microdrive includes four nichrome quadrupole tubes (14 mm; California Fine Wire Company), gold-plated (Neuralynx) with an impedance of 200 to 250 kU, arranged in one row (CA1 axis from CA3 on the dorsal hippocampus) And then transplanted to dorsal CA1 (AP, -1.8). The reference electrode was placed in a fiber tube on the hippocampus.

Surgery: Mice were anesthetized with avertin, restrained in a stereotactic instrument, and craniotomy was performed to expose the visual, somatosensory, and prefrontal cortex. The microdrive was implanted and slowly descended to the target depth. Mice were allowed to recover for a period of 4 days.

Electrophysiology in vivo: After a 2-3 day habituation period in the chamber, recording began with animals allowed to roam freely in a small open field. The recording session consisted of a period of 10-15 minutes followed by an additional 10 minutes with the animals exposed to the blinking LED immediately removing the sheet and immediately removing the sheet, with the LEDs blinking at 40 Hz but completely blocked with a black acrylic polypropylene sheet. Data was acquired using a Neuralynx SX system (Neuralynx, Bozeman, MT, USA) and signals were sampled at 32,556 Hz. Animals were tracked using a red light-emitting diode attached to a microdrive. In the results of the experiment, the mice were subjected to terminal anesthesia and the electrode positions were marked by electrolytic lesions of brain tissue with 50 μA current for 10 seconds individually through each electrode to confirm the anatomical position.

Spike: A single unit was manually separated by drawing cluster boundaries around the 3D protrusion of the recorded spike, presented in SpikEsort3D software (Neuralynx). Cells were considered as vertebral neurons when the average spike width was greater than 200 μs and the composite spike index (CSI)≧5.

Data analysis: LFP was first filtered to the Nyquist frequency of the target sampling rate and then downsampled by a factor of 20 (at 1628 Hz). Output spectral analysis was performed using the pwelch function in Matlab using a 500 ms time window with 50% overlap, and only segments of LFP data with animal speeds >4 cm/s were included in the analysis. A weighted phase retardation index (WPLI), a measure of interference that is more suitable for small rodent brains, was used as described above (Vinck et al., 2011) to counteract any potential contamination of interference by volume conducting signals. WPLI was calculated for electrode pairs in anatomically distinct regions only for periods of animal speed >4 cm/s. All analyzes were performed using Matlab.

Control of vertebral cells 40 Hz: The relationship between spike excitation frequency and 40 Hz LFP phase was calculated as described above using the Circular Statistics Toolbox (Middleton and McHugh, 2016). Briefly, spikes were sorted and LFP traces were filtered using a continuous wavelet transform with a cmor 1.5-1 wavelet centered at 40 Hz to return the instantaneous signal phase and amplitude. Spike time was linearly interpolated to determine the phase, and gamma floors and valleys were defined as 0 and 180 degrees, respectively. The resulting phase values were binned to generate excitation rates at 20-degree intervals. Cells were considered to be phase-locked only if they had a significantly different distribution (P<0.05 circular Rayleigh test) than the uniform, and the phase lock intensity was calculated as the average result length.

RNA sequencing

Isolation of microglia: The visual cortex was quickly dissected and placed in ice-cold Hanks' balanced salt solution (HBSS) (Gibco by Life Technologies, catalog number 14175-095). Tissues were enzymatically digested using a neural tissue separation kit (P) (Miltenyi Biotec, catalog number 130-092-628) with slight modifications according to the manufacturer's protocol. Specifically, tissues were enzymatically digested at 37° C. for 15 minutes instead of 35 minutes, and the resulting cell suspension was replaced with a 40 μm cell filter (Falcon Cell Strainers, Sterile, Corning, product 352340) instead of 70 μm MACS SmartStrainer. Passed. Subsequently, a CD11b mouse clone M1/70.15.11.5 (Miltenyi Biotec, 130-098-088) and a picoerythrin (PE) conjugated CD45 antibody (BD Pharmingen) conjugated with allopicocyanin (APC) according to the manufacturer's recommendations , 553081) to stain the resulting cell suspension. CD11b and CD45 positive microglia were then purified using FACS. Double lines were identified using the strict side scattering width vs. area and forward scattering width vs. area criteria of the standard and only a single line was gated. Viable cells were identified by staining with propidium iodide (PI) and gating only PI negative cells. CD11b and CD45 double positive EL were sorted into 1.5 ml centrifuge tubes containing 500 μl of RNA lysis buffer (QIAGEN, catalog number 74134) with 1% β-mercaptoethanol (Sigma-Aldrich, catalog number M6250). RNA was extracted using the RNeasy Plus Mini Kit (QIAGEN, catalog number 74134) according to the manufacturer's protocol. RNA was eluted and then stored at -80°C until full transcript amplification, library preparation and sequencing.

Isolation of neurons: The visual cortex was homogenized in 0.5 mL ice-cold PBS with protease inhibitor and the suspension was centrifuged at 1600 g for 10 minutes. The pellet was resuspended in 5 mL NF-1 hypotonic buffer, incubated for 5 minutes, then downs homogenized (Paste A) with 30 strokes. 5 mL NF-1 buffer was added to the suspension, and the pestle was washed with 10 ml NF-1 buffer to total 20 mL. All were collected in 50 conical tubes and the homogenate was filtered through a 40 μm mesh filter. Pellet nuclei for 15 minutes at 3,000 rpm (1,600 x g). Resuspended in 30 mL NF-1 buffer and mixed well. Rocked homogenate for 1 hour at 4°C. The pellet was washed once with 20 mL NF-1 buffer, centrifuged at 3,000 rpm for 15 minutes, and resuspended in 2-5 mL PBS + 1% BSA + protease inhibitor without touching the pellet on ice for 20 minutes. Alexa Fluor 488 or Alexa Fluor 647 conjugated NeuN antibody (1:500) was added to the tube and incubated at 4° C. for 20 minutes. Unbound antibody was washed by suspension and centrifugation with PBS + 1% BSA + protease inhibitor. The nuclei were spun down at 3,000 rpm for 15 minutes, resuspended in 0.5 mL (PBS+protease inhibitor) and filtered with a 40 μm mesh filter for FACS sorting. For nuclear gating, 2 drops of nuclear blue live preparation probe reagent were added (Thermo Fischer Scientific; Cat. No. R37605). NeuN double positive EL was sorted into 1.5 ml centrifuge tubes containing 500 μl of RNA lysis buffer (QIAGEN, catalog number 74134) with 1% β-mercaptoethanol (Sigma-Aldrich, catalog number M6250). RNA was extracted using the RNeasy Plus Mini Kit (QIAGEN, catalog number 74134) according to the manufacturer's protocol. RNA was eluted and then stored at -80°C until full transcript amplification, library preparation and sequencing.

RNA library preparation: The extracted total RNA was applied to QC using an advanced analytical-fragment analyzer before library preparation. SMARTer Stranded Total RNA-Seq Kit-Pico Input was used for P301S neurons, CK-p25 neurons and P301S microglia RNA-seq. SMART-Seq v4 Ultra Low Input RNA Kit was used for CK-p25 microglia specific RNA-seq. The library was prepared according to the manufacturer's instructions and sequenced against the Illumina Nextseq 500 platform at the MIT BioMicro Center.

Raw fastq data from a 40-bp paired sequence sequencing read was aligned to the mouse mm9 reference genome using STAR2.4. The total number of reads and percentage of aligned reads are as follows: CK-p25 neurons-total reads 18094674.857 ± 827050.464; Percentage sorted by 85.323 ±0.414. P301S neurons-total readings 22792713.18 ± 6308817.636; Percentage sorted by 84.45 ± 0.548. CK-p25 microglia-total readings 28694964.5 ± 435841.6674; Percentage sorted by 89.43 ± 0.25. [P301S microglia-total reading 18990369.2 ± 1667316.196; Percentage sorted by 27.243 ± 4.04. Due to this very low mapping rate, this experiment requires further consideration]. The mapped readings were processed by Cufflinks2.2 (Trapnell et al., 2012) using mm9 reference gene annotation to estimate the abundance of transcripts with library type as fr-second strand (for chain neuron data). Gene differential expression tests between groups were performed using the Cuffdiff module with p value <0.05 (for neurons). The geometric method was chosen as the library normalization method for Cuffdiff. For microglia data, the number of gene exons was quantified from non-stranded RNA-Seq data using the featureCounts tool, and statistical significance was calculated using DESeq2. Group FPKM values for differently expressed genes and other genes were visualized using a color coded scatter plot.

Z-scores of replication-expression FPKM values for genes that are expressed differently were visualized in heat maps for different sample groups. Gene ontology for microglia-specific DEG is performed using the Metascape tool, while neuron-specific DEG is performed using TOPPGENE.

Proteomics and phosphorus-protein physics

Sample preparation, reduction, alkylation and trypsin digestion: the visual cortex was dissected, rapidly frozen in liquid nitrogen and stored in a -80°C freezer until further use. The sample was then homogenized using a plastic hand-held motor driven homogenizer with a freshly prepared 8M urea solution. The protein concentration of the sample was quantified using Bio-Rad protein assay. Samples containing 1 mg of protein per ml were prepared, aliquoted and stored in a -80°C freezer until further use. The protein was reduced to 10 mM dithiothreitol (DTT) at 56° C. for 1 hour, alkylated with 50 mM iodoacetamide at room temperature (RT) for 1 hour, and less than 1 M with 100 mM ammonium acetic acid at pH 8.9. Diluted with urea. Proteins were digested using sequencing grade trypsin (Promega; 1 μg trypsin per 50 μg protein) overnight at RT. Enzyme activity was quenched by acidifying the sample with acetic acid. The peptide mixture was desalted, concentrated in a C18 Sep-Pak Plus cartridge (Waters) and eluted with 50% acetonitrile, 0.1% formic acid and 0.1% acetic acid. The solvent was evaporated in a SpeedVac vacuum centrifuge. Aliquots of 400 μg of each sample were aliquoted, frozen in liquid nitrogen for 5 minutes, and lyophilized at -80°C and stored.

TMT labeling: TMT labeling and phosphopeptide concentration: The lyophilized peptide was labeled with TMT-10-plex Mass Tag Labeling Kit (Thermo). For each TMT multiplex, a pooled sample consisting of a combination of equal amounts of peptides from a neurodegenerative WT, unstimulated and 40 Hz tuned mouse model was included, and relative quantification for normalized channels was determined. Made possible. For TMT labeling, aliquots of 9 mouse peptides at pH 8.5 and 9 samples from one normalization channel (400 μg peptide for each channel) are 100 μL of 70% (volume/volume) ethanol, 30% (volume) /Volume) Resuspended in 0.5 M triethyl-ammonium bicarbonate and incubated with TMT reagent resuspended in 40 μL dry acetonitrile for 1 hour at room temperature. Samples were concentrated, combined and concentrated to dryness using a SpeedVac vacuum centrifuge.

Peptide sorting: TMT-labeled peptide pellets were sorted via high-pH reverse phase HPLC. The peptide was resuspended in 100 uL Buffer A (10 mM TEAB, pH 8) and 4.6 mm using a 90 minute gradient with a flow rate of 1 minute/minute using Buffer B (90% MeCN, 10 mM TEAB, pH 8). x 250 mm 300Extend-C18, 5 um column (Agilent). The gradient is as follows: 1~5% B (0~10 min), 5~35% B (10~70 min), 35~70% B (70~80 min), 70% B (80~90 min) ). Fractions were collected over 75 minutes at 10-85 minute intervals. Fractions were linked non-consecutively into 15 fractions (1+16+31+46+61, 2+17+32+47+62, etc.). Thereafter, the fraction was almost dried by speed-vac (Thermo Scientific Savant).

Phosphopeptide Concentration: The phosphopeptide was concentrated from each of the 15 fractions using the High-Select Fe-NTA phosphopeptide enrichment kit (Thermo) according to the manufacturer's instructions.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS): Pre-column (home-made, 6 cm 10 μm C18) over 140 min gradient before nanoelectrospray using QExactive Plus mass spectrometer (Thermo) and self Peptides were separated by reverse phase HPLC (Thermo Easy nLC1000) using a Pack 5 μm tip analysis column (12 cm 5 μm C18, new object). Solvent A was 0.1% formic acid and solvent B was 80% MeCN/0.1% formic acid. The slope conditions are 0 to 10% B (0 to 5 minutes), 10 to 30% B (5 to 105 minutes), 30 to 40% B (105 to 119 minutes), and 40 to 60% B (119 to 124 minutes). , 60~100% B (124~126 min), 100% B (126~136 min), 100~0% B (136~138 min), 0% B (138~140 min), and mass spectrometer data It was operated in dependent mode. The parameters for the full scan MS were 70,000 resolution over 350-2000 m/z, AGC 3e6 and max IT 50 ms. The full MS scan was followed by MS/MS for the top 10 precursor ions in each cycle with an NCE of 34 and a dynamic exclusion of 30 seconds. Raw mass spectrum data files (.raw) were searched using Proteome Discoverer (Thermo) and Mascot version 2.4.1 (Matrix Science). Mascot search parameters include: 10 ppm mass tolerance for precursor ions; Fragment ion mass tolerance of 15 mmu; Two missing splits of trypsin; Fixed modifications of carbamidomethylation of cysteine and TMT 10plex modifications of lysine and peptide N-terminus; There are variable modifications that are methionine oxidation, tyrosine phosphorylation, and serine/threonine phosphorylation. TMT quantification was obtained using Proteome Discoverer and corrected to isotopes according to the manufacturer's instructions and normalized to the average of each TMT channel. Only peptides with Mascot scores greater than or equal to 25 and isolation interference less than or equal to 30 were included in the data analysis.

The washed preparative column was packed with an inner packaging analysis capillary column (50 μm ID Х 12 cm (filled with 5 μm C18 beads) with electrospray tip (approximately 1 μm orifice) (YMC gel, ODSAQ, 12 nm, S-5) μm, AQ12S05)] in series. The peptide is eluted using a gradient of 9 to 70% acetonitrile in 0.2 M acetic acid at a flow rate of 0.2 ml/min with a flow fractionation of about 10,000:1 using a gradient of acetonitrile of 140 to 90 minutes (phosphopeptide) or 90 minutes (total peptide) The final electrospray flow rate of about 20 nL/min was calculated. A total of 15 fractions were collected from each sample. Thermo Q Exactive Hybrid Quadrupole-Orbitrap Plus mass with the following settings (spray voltage, 2 kV; no sheath or auxiliary gas flow, heated capillary temperature, 250°C; 50% S-lens radio frequency level) The phosphopeptide was analyzed using an analyzer. Q Exactive was run in a data-dependent acquisition mode. Full scan MS spectrum [mass/charge ratio (m/z), 350-2000; Resolution at m/z 200, 70,000] was detected in the Orbitrap analyzer following accumulation of ions at the 3e6 target value based on the predicted AGC from the previous scan. For every scan, the 15 strongest ions are isolated (higher-energy collisional dissociation (HCD)) with an implantation time of up to 300 ms and 35,000 resolution (isolated width of 0.4 m/z) and fragmented ( Impact energy (CE): 32%). Total peptide analysis was performed on an LTQ Orbitrap XL mass spectrometer with the following settings (spray voltage, 2 kV; no sheath or auxiliary gas flow, heated capillary temperature, 250° C. Analysis was performed in a data dependent acquisition mode. , The full scan mass spectrum (m/z range 400-2000, resolution 60,000) was detected on an Orbitrap analyzer (ion target value 5x105), for each full scan, the 10 strongest ions were HCD in HCD cells (CE: 75%) It was isolated (3 Da width) and fragmented by iTRAQ marker and detected by Orbitrap (ion target value 1x105) for quantification of ion.

Mass spectrometry peptide mapping data analysis: Raw mass spectrum data files were loaded into Proteome Discoverer version 1.4.1.14 (DB version: 79) (Thermo) and searched against the mouse SwissProt database using Mascot version 2.4 (Matrix Science). TMT reporter quantification was extracted and isotopes were modified in Proteome Discoverer. The tandem mass spectrum was consistent with an initial mass tolerance of 10 ppm for the precursor mass and 15 mmu for fragment ions. Cysteine carbamidomethylation, TMT-labeled lysine and protein N-terminus were searched as fixed modifications. The phosphorylation of oxidized methionine and serine, threonine and tyrosine was searched as variable modifications. The minimum peptide length was 7 amino acids. The data set was sorted with an ion score of greater than 20 for the entire peptide to ensure high confidence in peptide identification and phosphorylation localization and achieved (FDR) below 1% for the peptide. Phosphopeptide quantification was normalized based on the intermediate relative peptide quantification obtained from the crude peptide analysis to correct for slight changes in sample amount between TMT-channels. For each phosphopeptide, relative quantification was expressed as the ratio between the TMT ionic strength from each analyzed sample and the included normalization channel.

Bioinformatics analysis: To discriminately identify peptides expressed and phosphorylated at significantly controlled ratios, we selected a random cutoff of ±20% difference with a modified P value of <0.05. The unregulated background pool consisted of peptides with a ratio of 0.8-1.2. Thus, subsequent bioinformatics analysis included peptides with a ratio of <0.8 and >1.2 compared to their normalized channels, which are considered down-regulated and up-regulated, respectively. The name of the protein from the protein accession number was converted to a gene list using the Uniprot ID mapping search tool. Protein networks were obtained by using the STRING database (version 10.5). All valid interaction sources except text mining were included to ensure high reliability, and a reliability score of 0.9 or higher was required. Gene Ontology (GO) term enrichment analysis is first performed using bioinformatics resources STRING (http://string-db.org), TOPPGENE (toppgene.cchmc.org) and Metascape (http://metascape.org). Process-related terms were performed, and then manually filtered to find terms that generally existed from these three resources. Reported GO terms obtained from TOPPGENE. For each individual mouse strain (C57BL6/J, CK-p25, P301S tau-tg and 5XFAD), the assay was derived from each pool of differently adjusted total peptides or S/T phosphopeptides (up and down adjusted). Gene set was included.

behavior

Open Field: Mice were placed in an open field box (dimension: length = 460 mm, width = 460 mm, and height = 400 mm; TSE-system), along with time measured as spent in the center and surrounding areas of the arena 5 Followed by Noldus (Ethovision) for min.

Increased cruciform maze (EPM): Mice were placed in the central area of the EPM (arbitrary maze dimension: arm length = 35 cm, width = 5 cm) and tracked using the Noldus program for 10 minutes. The time spent in each arm and center of the EPM was calculated offline.

Recognition of new objects in CK-p25 and P301S tau mice: Place the mouse in an open field arena and calculate the time spent in the center of the arena. The next day, the mouse was put back into the same open field box that now contains two existing objects (new objects or you'll get used to it in the next session), and you were asked to explore them for 10 minutes. Next, it returned to the same arena 10 minutes after the last search, and one of the two objects was replaced with a new one. Mouse behavior was monitored for 7 minutes. The time spent exploring both existing and new objects was recorded using Noldus and calculated offline.

Seizure sensitivity: Mice were injected with picrotoxin (i.p injection), placed in an open field arena and recorded for 30 minutes using Noldus and also a side mounted camera. The distance traveled was calculated offline by Noldus, and the severity of seizures was manually scored.

Morris Underwater Maze: MWM is a test that evaluates spatial learning. MWM relies on visual cues to search for a submerged escape platform by searching at the starting point around the open swimming arena. The device consisted of a circular paste (122 cm in diameter) filled with tap water (22° C. to 24° C.) and the solution was made opaque by adding a non-toxic white paint. An escape platform (10 cm in diameter) with blunt protruding corners for a better grip is placed 1 inch below the water level. The pool was divided into four equal quadrants labeled N (north), E (east), S (south) and W (west). Mice were placed in a maze in random order across the assay, from the edge, for a 60 second assay. The time required to find the hidden platform (wait time) was recorded. The probe test was performed 24 hours after the last training assay with the submerged platform removed. All training and probe assay tests were recorded using Noldus.

CK-p25, P301S and 5XFAD mice were tested in MWM in the afternoon (3-7pm), where they experienced GENUS in the morning (up to 12pm) and limited the behavioral test time to 4 hours per day. We used at least twice and up to four tests per day during training to ensure that we were able to implement all groups in MWM while also maintaining a strict cancer/light cycle. Despite the difference in training days, all AD mice used in MWM performed a similar number of total training courses.

Statistical analysis

Statistical analysis was performed in SPSS, Matlab or Prism. The sample size was not predetermined. Statistical significance was calculated using an independent sample t-test, Wilcoxon Rank-sum test, one-way ANOVA or Bonferroni post-hoc analysis and two-way repeat measurement ANOVA with Krushkal Wallis test. Became. Statistical significance was set to 0.05.

conclusion

Aspects of the invention of the present disclosure relate to each individual feature, system, article, material, kit and/or method described herein. In addition, any combination of two or more of these features, systems, articles, materials, kits and/or methods, if the features, systems, articles, materials, kits and/or methods are inconsistent with each other, is the scope of the invention of the present disclosure. Is included within.

In addition, various inventive concepts may be implemented as one or more methods, one of which is provided as an example. Operations performed as part of the method can be ordered in any suitable way. Thus, implementations may be configured to perform operations in a different order than illustrated, and may include performing some operations simultaneously, even though they appear as sequential operations in the exemplary implementation.

All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

It is to be understood that all definitions defined and used herein control dictionary definitions, definitions in documents incorporated by reference and/or the general meaning of the defined terms.

As used herein, the terms “about” and “approximately” when used in conjunction with numerical values and/or ranges, generally refer to numerical values and/or ranges close to the numerical values and/or ranges recited. In some cases, the terms “about” and “approximately” can mean within ±10% of the value quoted (including the quoted value itself). For example, in some cases, “about 100 [units]” may mean within ± 10% of 100 (eg, 90 to 110). In other cases, the terms “about” and “approximately” may mean within ±5% of the value quoted (including the quoted value itself). In another case, the terms “about” and “approximately” can mean within ±1% of the value quoted (including the quoted value itself). The terms "about" and "approximately" may be used interchangeably.

As used herein, indefinite articles ("a" and "an") should be understood as meaning "at least one", unless expressly indicated otherwise.

As used herein, the phrase “and/or”, within this specification and claims, is conjugated, that is, in some cases, in combination, and in other cases, separately present, either or both of the elements. It should be understood as meaning ". Multiple elements listed as “and/or” should be interpreted in the same way, ie “one or more” of the joined elements. Elements other than those specifically identified by the "and/or" clause, other elements that are related to or not related to the specifically identified element may optionally be present. Thus, as a non-limiting example, when used with an open language such as "comprising", a reference to "A and/or B" is: in one embodiment only A (optionally including elements other than B); In other embodiments, only B (optionally including elements other than A); In another embodiment both A and B (optionally including other elements); And the like.

As used herein in this specification and claims, it should be understood that “or” has the same meaning as “and/or” as defined above. For example, when separating items from a list, “or” or “and/or” is inclusive, that is, a list of numbers or elements that includes at least one, but which contains more than one, and, optionally, an additional list. It should also be construed to include missing articles. Conversely, only clearly indicated terms, such as “consisting of,” when used in the claims, such as “only one” or “exactly one”, or claims, include exactly one element in a list of numbers or elements. Will reference. In general, as used herein, the term “or” is an exclusive alternative when an exclusive term precedes, such as “any one,” “one of,” “only one,” or “exactly one”. (Ie, “one or the other, but not both”). When used in the claims, "consisting essentially of" will have its ordinary meaning as used in the field of patent law.

As used herein in this specification and claims, the phrase “at least one” with respect to a list of one or more elements means at least one element selected from any one or more elements in the element list, but the element list Each element specifically listed in and at least one of all elements must be included, and it is not necessary to exclude any combination of elements in the element list. This definition also allows the presence of the element at least in the list of elements referred to by the phrase "at least one," whether or not the corresponding element is related to the specifically identified element in addition to the specifically identified element. Thus, by way of non-limiting example, "at least one of A and B" (or equivalently, "at least one of A or B," or, equally, "at least one of A and/or B") is: one embodiment In, without B, at least one A, optionally two or more (and optionally including elements other than B); In other embodiments, without A, at least one B, optionally two or more (and optionally including elements other than A); In another embodiment, at least one A, optionally two or more, and at least one B, optionally two or more (and optionally including other elements); And the like.

As in the above specification, in the claims "comprising/including", "carrying/having", "containing", "involving", "holding" It should be understood that transitional phrases such as ", "composed of", etc., are meant to be open-ended, ie including but not limited to. Only the transitional phrases “consisting of” and “consisting essentially of” correspond to closed or semi-closed transitions, as described in the United States Patent and Trademark Office patent examination procedure manual 2111.03.

Claims (86)

A method of treating dementia or Alzheimer's disease in a subject in need thereof, the method comprising
A) Chronic visual stimulation with a frequency of about 30 Hz to about 50 Hz to the subject to entrain synchronized gamma vibration in multiple brain regions of the subject, including at least the prefrontal cortex (PFC) and hippocampus of the subject. A method comprising non-invasive delivery. The method of claim 1, wherein in A), the chronic visual stimulus has a frequency of about 35 Hz to about 45 Hz. The method of claim 2, wherein in A), the chronic visual stimulus has a frequency of about 40 Hz. The method of claim 3, wherein A) comprises inducing a local field translocation (LFP) of about 40 Hz in at least the prefrontal cortex and hippocampus of the subject. The method of claim 2, wherein in A), the chronic visual stimulus has a 50% duty cycle. 3. The method of claim 2, wherein A) comprises driving a light emitting diode (LED) array with a square wave current signal to produce a chronic visual stimulus. The method of claim 6,
The square wave current signal has a 50% duty cycle;
The method of which the frequency of the square wave current signal is 40 Hz or approximately 40 Hz. The method of claim 7, wherein A) comprises inducing a local field translocation (LFP) of about 40 Hz in at least the prefrontal cortex and hippocampus of the subject. The method of claim 2, wherein A) comprises non-invasively delivering a chronic visual stimulus at least 1 hour per day for more than 7 days. The method of claim 9, wherein A) comprises non-invasively delivering chronic visual stimulation at least 1 hour per day for at least 22 days. The method of claim 10, wherein A) comprises non-invasively delivering chronic visual stimulation at least 1 hour per day for at least 42 days. The method of claim 10,
A) includes driving a light emitting diode (LED) array with a square wave current signal to create a chronic visual stimulus;
The square wave current signal has a 50% duty cycle;
The method of which the frequency of the square wave current signal is 40 Hz or approximately 40 Hz. The method of claim 12, wherein A) comprises inducing a local field translocation (LFP) of about 40 Hz in at least the prefrontal cortex and hippocampus of the subject. The method of claim 13, wherein the multiple brain regions of the subject include the subject's visual cortex, somatosensory cortex, hippocampus and prefrontal cortex. According to claim 1, A) is
A1) non-invasively delivering chronic visual stimulation with a frequency of about 30 Hz to about 50 Hz to the subject to simultaneously synchronize synchronized gamma vibrations in multiple brain regions of the subject, including at least the prefrontal cortex and hippocampus of the subject A method comprising steps. The method of claim 15, A1) is
A method comprising significantly increasing gamma interference with a frequency of 30 Hz to 50 Hz between a plurality of brain regions of a subject, including at least the prefrontal cortex and hippocampus of the subject. The method of claim 16, wherein the multiple brain regions of the subject include the subject's visual cortex, somatosensory cortex, hippocampus and prefrontal cortex. The method of claim 17, wherein in A), the chronic visual stimulus has a frequency from about 35 Hz to about 45 Hz. The method of claim 18, wherein in A), the chronic visual stimulus has a frequency of about 40 Hz. 17. The method of claim 16, wherein A) comprises non-invasively delivering chronic visual stimulation at least 1 hour per day for more than 7 days. 21. The method of claim 20, wherein A) comprises non-invasively delivering chronic visual stimulation at least 1 hour per day for at least 22 days. The method of claim 21, wherein A) comprises non-invasively delivering chronic visual stimulation at least 1 hour per day for at least 42 days. The method of claim 22,
A) includes driving a light emitting diode (LED) array with a square wave current signal to create a chronic visual stimulus;
The square wave current signal has a 50% duty cycle;
The method of the frequency of the square wave current signal is 40 Hz or approximately 40 Hz. The method of claim 15, A1) is
A2) further non-invasively delivering a chronic visual stimulus having a frequency of about 30 Hz to about 50 Hz to modulate neuronal activity between the subject's multiple brain regions, including at least the prefrontal cortex and hippocampus of the subject. Included, Method. The method of claim 24, A2) is
Non-invasively delivering a chronic visual stimulus having a frequency of about 30 Hz to about 50 Hz to modulate neuronal activity between a plurality of brain regions of the subject, including at least the prefrontal cortex and hippocampus of the subject. , Way. The method of claim 25, wherein the multiple brain regions of the subject include the subject's visual cortex, somatosensory cortex, hippocampus and prefrontal cortex. 27. The method of claim 26, wherein in A), the chronic visual stimulus has a frequency of about 35 Hz to about 45 Hz. 28. The method of claim 27, wherein in A), the chronic visual stimulus has a frequency of about 40 Hz. 25. The method of claim 24, wherein A) comprises non-invasively delivering a chronic visual stimulus at least 1 hour per day for more than 7 days. 26. The method of claim 25, wherein A) comprises non-invasively delivering chronic visual stimulation at least 1 hour per day for at least 22 days. 27. The method of claim 26, wherein A) comprises the non-invasive delivery of chronic visual stimulation by at least 1 hour per day for at least 42 days. The method of claim 27,
A) includes driving a light emitting diode (LED) array with a square wave current signal to create a chronic visual stimulus;
The square wave current signal has a 50% duty cycle;
The method of which the frequency of the square wave current signal is 40 Hz or approximately 40 Hz. The method of claim 24, A2) is
A3) further comprising non-invasively delivering a chronic visual stimulus having a frequency of about 30 Hz to about 50 Hz to reduce neurodegeneration in multiple brain regions of the subject, including at least the prefrontal cortex and hippocampus of the subject. How to. The method of claim 33, A3) is
Non-invasively delivering a chronic visual stimulus having a frequency of about 30 Hz to about 50 Hz to reduce amyloid plaque in multiple brain regions of the subject, including at least the prefrontal cortex and hippocampus of the subject, Way. The method of claim 33, A3) is
Non-invasively delivering a chronic visual stimulus having a frequency of about 30 Hz to about 50 Hz to reduce tau hyperphosphorylation in multiple brain regions of the subject, including at least the prefrontal cortex and hippocampus of the subject. , Way. The method of claim 33, A3) is
Non-invasively delivering a chronic visual stimulus having a frequency of about 30 Hz to about 50 Hz to reduce loss of neurons and synapses in multiple brain regions of the subject, including at least the prefrontal cortex and hippocampus of the subject. Included, Method. The method of claim 33, A3) is
Non-invasively delivering a chronic visual stimulus having a frequency of about 30 Hz to about 50 Hz to reduce brain atrophy in multiple brain regions of the subject, including at least the prefrontal cortex and hippocampus of the subject, Way. The method of claim 33, A3) is
Non-invasively delivering a chronic visual stimulus having a frequency of about 30 Hz to about 50 Hz to reduce ventricular dilatation in multiple brain regions of the subject, including at least the prefrontal cortex and hippocampus of the subject, Way. 34. The method of claim 33, wherein A) comprises non-invasively delivering a chronic visual stimulus for at least 1 hour per day for more than 7 days. 40. The method of claim 39, wherein A) comprises non-invasively delivering chronic visual stimulation at least 1 hour per day for at least 22 days. 41. The method of claim 40, wherein A) comprises non-invasively delivering a chronic visual stimulus by at least 1 hour per day for at least 42 days. The method of claim 41
A) includes driving a light emitting diode (LED) array with a square wave current signal to create a chronic visual stimulus;
The square wave current signal has a 50% duty cycle;
The method of the frequency of the square wave current signal is 40 Hz or approximately 40 Hz. 39. The method of any one of claims 34-38, wherein A) comprises non-invasively delivering chronic visual stimulation at least 1 hour per day for more than 7 days. 44. The method of claim 43, wherein A) comprises non-invasively delivering chronic visual stimulation by at least 1 hour per day for at least 22 days. 45. The method of claim 44, wherein A) comprises non-invasively delivering chronic visual stimulation at least 1 hour per day for at least 42 days. The method of claim 45,
A) includes driving a light emitting diode (LED) array with a square wave current signal to create a chronic visual stimulus;
The square wave current signal has a 50% duty cycle;
The method of the frequency of the square wave current signal is 40 Hz or approximately 40 Hz. The method of claim 24, A2) is
A3) further comprising non-invasively delivering a chronic visual stimulus having a frequency of about 30 Hz to about 50 Hz to reduce neuroinflammation in multiple brain regions of the subject, including at least the prefrontal cortex and hippocampus of the subject. How to. The method of claim 47, A3) is
A4) non-invasively delivering chronic visual stimulation with a frequency of about 30 Hz to about 50 Hz to reduce the immune response of at least some microglia in a number of brain regions of the subject, including at least the prefrontal cortex and hippocampus of the subject The method further comprising a step. The method of claim 48, A4) is
Non-invasively delivering a chronic visual stimulus having a frequency of about 30 Hz to about 50 Hz to morphologically modify at least some microglia in multiple brain regions of the subject, including at least the prefrontal cortex and hippocampus of the subject; Method further included. The method of claim 48, A4) is
Non-invasively delivering a chronic visual stimulus with a frequency of about 30 Hz to about 50 Hz to increase proteolysis in at least some microglia in multiple brain regions of the subject, including at least the prefrontal cortex and hippocampus of the subject Method further included. 49. The method of claim 48, wherein A) comprises non-invasively delivering chronic visual stimulation at least 1 hour per day for more than 7 days. The method of claim 51, wherein A) comprises non-invasively delivering chronic visual stimulation at least 1 hour per day for at least 22 days. 53. The method of claim 52, wherein A) comprises non-invasively delivering chronic visual stimulation at least 1 hour per day for at least 42 days. The method of claim 53,
A) includes driving a light emitting diode (LED) array with a square wave current signal to create a chronic visual stimulus;
The square wave current signal has a 50% duty cycle;
The method of which the frequency of the square wave current signal is 40 Hz or approximately 40 Hz. The method of claim 24, A2) is
Abnormally modified associated with at least one of membrane trafficking, intracellular transport, synaptic function, neuroinflammatory, apoptosis processes, and DNA damage in multiple brain regions of the subject, including at least the prefrontal cortex and hippocampus of the subject The method further comprises non-invasively delivering a chronic visual stimulus having a frequency of about 30 Hz to about 50 Hz to improve genes and proteins. The method of claim 15, A1) is
A2) A method further comprising non-invasively delivering a chronic visual stimulus having a frequency of about 30 Hz to about 50 Hz to improve the subject's learning and memory. 57. The method of claim 56, wherein A) comprises non-invasively delivering a chronic visual stimulus for at least 1 hour per day for more than 7 days. The method of claim 57, wherein A) comprises non-invasively delivering chronic visual stimulation at least 1 hour per day for at least 22 days. 59. The method of claim 58, wherein A) comprises non-invasively delivering chronic visual stimulation at least 1 hour per day for at least 42 days. The method of claim 59,
A) includes driving a light emitting diode (LED) array with a square wave current signal to create a chronic visual stimulus;
The square wave current signal has a 50% duty cycle;
The method of which the frequency of the square wave current signal is 40 Hz or approximately 40 Hz. A method of treating dementia or Alzheimer's disease in a subject in need thereof, the method comprising
A) non-invasively chronic chronic stimulation with a frequency of about 30 Hz to about 50 Hz to the subject to tune synchronized gamma vibrations in multiple brain regions of the subject, including at least the prefrontal cortex (PFC) and hippocampus of the subject Transferring steps include: A):
A1) synchronized synchronized gamma vibrations in multiple brain regions of the subject, including at least the prefrontal cortex and hippocampus of the subject;
A2) modulates neuronal activity between multiple brain regions of the subject, including at least the prefrontal cortex and hippocampus of the subject;
A3) reducing neurodegeneration in multiple brain regions of the subject, including at least the prefrontal cortex and hippocampus of the subject;
A4) reducing neuroinflammation in multiple brain regions of the subject, including at least the prefrontal cortex and hippocampus of the subject;
A5) Abnormally modified genes associated with at least one of membrane transport, intracellular transport, synaptic function, neuroinflammatory, apoptosis processes, and DNA damage in multiple brain regions of the subject, including at least the prefrontal cortex and hippocampus of the subject, and Improve protein;
A5) A method of non-invasive delivery of a chronic visual stimulus having a frequency of about 30 Hz to about 50 Hz to a subject to improve learning and memory of the subject. The method of claim 61, wherein A) comprises inducing a local field translocation (LFP) of about 40 Hz in at least the prefrontal cortex and hippocampus of the subject. 63. The method of claim 62, wherein A) comprises non-invasively delivering chronic visual stimulation at least 1 hour per day for at least 22 days. 64. The method of claim 63, wherein A) comprises non-invasively delivering chronic visual stimulation by at least 1 hour per day for at least 42 days. The method of claim 63,
A) includes driving a light emitting diode (LED) array with a square wave current signal to create a chronic visual stimulus;
The square wave current signal has a 50% duty cycle;
The method of which the frequency of the square wave current signal is 40 Hz or approximately 40 Hz. The method of claim 61,
A) includes driving a light emitting diode (LED) array with a square wave current signal to create a chronic visual stimulus;
The square wave current signal has a 50% duty cycle;
The method of which the frequency of the square wave current signal is 40 Hz or approximately 40 Hz. A method of treating dementia or Alzheimer's disease in a subject in need thereof, the method comprising
A) Frequency of about 30 Hz to about 50 Hz to the subject to tune synchronized gamma vibrations in multiple brain regions of the subject and to improve abnormally modified genes and proteins in neurons regressing in multiple brain regions of the subject And non-invasively delivering a chronic visual stimulus having a method. 68. The abnormally modified gene and protein in neurons that degenerate in multiple brain regions of a subject is associated with at least one of membrane transport, intracellular transport, synaptic function, neuroinflammatory, apoptosis processes, and DNA damage. , Way. The method of claim 68, wherein the abnormally modified gene and protein comprises a gene derived from a NueN-positive neuronucleus in at least the visual cortex of a subject. The method of claim 69, wherein A) comprises non-invasively delivering chronic visual stimulation to reduce or maintain a percentage of NueN-positive neuronuclei in at least the visual cortex of the subject. 69. The method of claim 68, wherein the abnormally modified gene and protein comprises S/T- phosphorylated protein, A) non-invasively delivering chronic visual stimulation to reduce S/T- phosphorylation in at least the visual cortex of the subject. A method comprising the step of modifying the S/T-phosphorylated protein. The method of claim 71, wherein the S/T-phosphorylated protein comprises dynamin1 (DNM-1), A) non-invasively delivering chronic visual stimulation to reduce Ser774 phosphorylation in DNM-1. How to include. 69. The abnormally modified gene and protein of claim 68, comprises vesicular glutamic transporter 1 (vGlut1) with significantly reduced puncta, and A) displays vGlut1 spots in multiple brain regions of the subject. A method comprising non-invasive delivery of a chronic visual stimulus to significantly increase. The method of claim 73, wherein the plurality of brain regions of the subject comprises the subject's visual cortex, somatosensory cortex, hippocampus and prefrontal cortex. 68. The method of claim 67, wherein A) comprises non-invasively delivering a chronic visual stimulus at least 1 hour per day for more than 7 days. The method of claim 75, wherein A) comprises non-invasively delivering chronic visual stimulation at least 1 hour per day for at least 22 days. 77. The method of claim 76, wherein A) comprises non-invasively delivering chronic visual stimulation by at least 1 hour per day for at least 42 days. The method of claim 77,
A) includes driving a light emitting diode (LED) array with a square wave current signal to create a chronic visual stimulus;
The square wave current signal has a 50% duty cycle;
The method of which the frequency of the square wave current signal is 40 Hz or approximately 40 Hz. A method of treating dementia or Alzheimer's disease in a subject in need thereof, the method comprising
A) Non-invasive delivery of chronic visual stimuli with frequencies between about 30 Hz and about 50 Hz to subjects to significantly increase gamma interference with frequencies between 30 Hz and 50 Hz between multiple brain regions of subjects And simultaneously synchronizing synchronized gamma vibrations in multiple brain regions of the subject. The method of claim 79, wherein the multiple brain regions of the subject include the subject's visual cortex, somatosensory cortex, hippocampus and prefrontal cortex. 81. The method of claim 80, wherein A) comprises inducing a local field translocation (LFP) of about 40 Hz in at least the prefrontal cortex and hippocampus of the subject. The method of claim 79, wherein A) comprises non-invasively delivering chronic visual stimulation at least 1 hour per day for more than 7 days. 83. The method of claim 82, wherein A) comprises non-invasively delivering chronic visual stimulation at least 1 hour per day for at least 22 days. 84. The method of claim 83, wherein A) comprises non-invasively delivering chronic visual stimulation by at least 1 hour per day for at least 42 days. The method of claim 84,
A) includes driving a light emitting diode (LED) array with a square wave current signal to create a chronic visual stimulus;
The square wave current signal has a 50% duty cycle;
The method of the frequency of the square wave current signal is 40 Hz or approximately 40 Hz. The method of claim 85, wherein A1) is
The method further comprising non-invasively delivering a chronic visual stimulus having a frequency of about 30 Hz to about 50 Hz to modulate neuronal activity between multiple brain regions of the subject.

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