Master EMN-Online 2021

Introduction to basic aspects of neurodegenerative diseases

Christian Gestreau - Associate Professor, Aix-Marseille University

Chapter 3: Neuroprotection as a strategy to act on

pathophysiological degenerative processes and to stop
neurodegenerative disease evolution: myth or clinical reality?

Introductory remarks

Regarding neurodegenerative diseases it is speculated that a treatment based on the etiology of the
disease can interfere with the pathological process and therefore can slow the evolution of the disease and
ultimately stop the disease.

Such a neuroprotective strategy is not yet available in clinic to treat patients suffering neurodegenerative
diseases. However, numerous experimental data have been obtained in animal models suggesting that
such a strategy is obviously possible for the future. But at present clinical data based on such proposed
strategies have shown really disappointing results.

The reasons of such negative effects in clinic are probably multiples. However it is really paradoxical
showing very promising experimental data in preclinic studies and such limited clinical effects. Possibly
animal models are not good enough to exactly reproduce the diseases, which contributes to limit the
transposition of data in patients. Moreover putative neuroprotective strategies are based on concepts
coming from our present knowledge of the basic mechanisms of the diseases, which are still very limited in
humans. But advances in understanding pathophysiological processes would contribute to better
understanding the molecular basis of the disease and further to propose new targets for innovative
strategies.

One of the most important limiting factors to promote neuroprotective treatments in patients, however, is
the true difficulty to evaluate clinical benefit of such treatments if they exist. Because neurodegenerative
diseases are defined by their chronicity and very long term developments correlated to very slow
pathological processes, because also of their heterogeneity and the fact that during the evaluation of
neuroprotective treatments patients continue to be administered with symptomatic treatments when
available, and because we have only very rare biological markers of the diseases, it is obviously difficult
and even impossible to evaluate the putative benefit of a given neuroprotective treatment.

So, the only possibility for that is to have a very long term follow up of large cohorts of patients to expect
detected some inflexion in the evolution of the disease compared to patient without such putative
protective treatment. Finally one major limiting factor is the total cost of such an evaluation, which is
difficult to support for most of the big pharmaceutical companies. In the present state of the art, the only
“neuroprotective” indication for a drug is related to the action of a drug named “riluzole” for treating
ALS. But the clinical benefit for patients is still very limited.

What means “neuroprotection”?

Clinicians and biologists certainly have different definitions of what neuroprotection could be. For
clinicians neuroprotection is rather a broad concept primarily related to the evaluation of the clinical
signs of a given disease for a given patient and of their evolution overtime. Clinicians are therefore
looking at some inflexion of the natural evolution of the disease under the influence of the
treatment. All changes in the slope of such an evolution are therefore considered as a “gain” for the
patient and are considered to correspond to a positive influence of the putative neuroprotective
agent against the natural course of the pathological process.

Such a benefit could be illustrated by a simple delay in the appearance of a new clinical sign
associated to aggravation of the pathology further interpreted as a gain for the patient (this could be
the case for the treatment of Alzheimer’s disease using anti-cholinesterasic agents which prolong the
autonomy of patients, for example) and ultimately by a total remission of the disease in a curative
dimension or at least in a stabilisation of the patient clinical state without any more aggravation. But
because of the heterogeneity of the diseases such appreciation is very difficult.

Regarding the biologist view of neuroprotection the situation is rather different and more precisely
focused on cellular processes. Neuroprotection for biologist means a way to protect the cells in
general and neurons in particular against a process tending to alter cell function and survival. Thus, a
neuroprotective action has to be focused on neuron survival and the exact term more appropriate to
the view of biologist is “neuro-cyto-protection”. In this respect studies are aimed at evaluating the
consequence of an aggressive strategy for the cell and possible rescue linked to application of a
compound the role of which will be to limit the cell destruction.

For example in standardized animal models of stroke the amplitude of lesion examined at cellular
level could be modified by appropriate agent limiting the cell death. But in such a case it is worth
mentioning that studying the behavioural consequences of experimental stroke and subsequent
neuroprotective treatment contributes to evaluate functional impact of the treatment. And in this
way the view of biologist crosses the interest of the clinician.

Consequently an efficient neuroprotective action as to be translated in a cell protection but also in a

functional improvement compared to natural evolution of a given disease. Of course because the
lessons are focused on cellular bases of neurodegenerative disease the next considerations will be
focused on these cellular approaches of the diseases. In this respect neuroprotection is based on
interaction with pathological processes although we have plenty limitations in understanding
currently what happens in the brain of patients.

Apoptosis, as a general mechanism of neuronal death in neurodegenerative

diseases.A target for neuroprotective strategies?
Neuronal death use two main mechanisms called “necrosis” and “apoptosis”. Autophagia is another
mechanism but this process could also correspond to an ultimate phase of apoptosis.

Necrosis is more likely related to stroke or to brain trauma. Necrosis is related to acute lesion in
which the cell death is a rapid process reversible in nature if treated rapidly. The main feature of the
process is a rather tardive loss in cell adhesion properties. Conversely, turgescence of cytosol is rapid
and caryolyse involves enzymatic processes. Fragmentation of DNA is hazardous. As previously

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mentioned (see Part I) inflammatory processes are concomitant of neuronal death and a glial scar
contributes to limit further possible re-innervation processes.

Apoptosis is a more physiological process, which contributes to biological development in its early
phases. For example, it contributes to selection procedures of neuronal pathway organization during
development but also in processes leading to the separation of fingers in the hand of primates.
Apoptosis is an irreversible process but it can be regulated or even prevented for example by using
some neurotrophic factors in very specific situations. In such a situation adhesion processes are lost
very rapidly whereas the turgescence of the cytosol is a rather tardive event. Apoptosis is
characterized by a major chromatin compaction and DNA fragmentation evidencing inter-
nucleosomes segments. The cell is forming what are called “apoptotic bodies” and
phagocytoseconstitutes one of the late events of apoptosis. When phagocytose is by the cell itself it
is called autophagia. Inflammatory processes are rather limited in case of apoptosis and no clear scar
is present after the cell death. Apoptosis is in fact a process evidenced as early as the end of the
XIXth century. Interestingly, whereas an increased apoptosis result in muscle atrophy for example,
conversely an inhibition of apoptosis results in a tumour formation.

Apoptosis is the current mechanism related to neurodegenerative diseases although in autopsy

samples the number of apoptosis features is limited, which is normal considering that
neurodegenerative processes mainly occur during the life of patients before death.

Among the most recent advances in apoptosis understanding it is worth mentioning the possibility to
finely regulate the molecular process. Internal as well as external cellular signals have been shown to
contribute to apoptosis regulatory processes. External cellular signals are for example represented by
trophic factors, hormones, cytokines or even environmental toxins which can contribute to transfer
external information into a given cell through a trans-membrane action. In that situation regulation
can facilitate or conversely inhibit apoptosis.

The cell itself is able to generate internal signals, for example in response to a cellular stress. In such
a situation it can be considered that the cell contribute to a form of suicide because of the intensity
of the stress. Certain glucocorticoid nuclear receptors as well as cell irradiation, some viral infections
or even transient hypoxia or dramatic increase in intracellular calcium ion concentrations are indeed
able to induce apoptosis through intracellular signals identified for example as poly ADP ribose
polymerase known as “PPAR signal”.

Molecular mechanisms of apoptosis have been described first during the year 1990 by Hortiz who
described the molecular basis of the so-called “programmed cell death” (Nobel Prize 2002). Working
on the nematode c.elegans animal model, Hortiz described the basis of the cell death as
corresponding to the action of specific genes called “killer genes”. The main genes were named Ced3
and Ced4 (Ced for cell death), the activation of which was demonstrated to induce apoptosis of the
cell. Gene activation was proposed to start only for starting apoptosis suggesting that such genes are
“non-adaptive “in nature, which means that they are “silent” all along the life and become active
only at the moment of the cell death.

Another major concept from the work by Horvitz was the description of a regulatory process of Ced3
and Ced4 gene expression involving another gene called Ced9. Ced9 was presented as a “protective”
gene against apoptosis acting as a pleiotropic antagonist gene, which means that it contributes all
the lifelong to “protect” the cell from the action of the killer genes by inhibiting permanently there
action. Thus Ced9 was proposed to permanently inhibit the action of Ced3 and Ced4. Consequently,
the release of the action of Ced9 is considered as a signal leading to the beginning of cell death.

Proteins encoded by genes Ced3 and Ced4 in c. elegans have a high level of homology with proteins
corresponding to the proteases “interleukin conversion enzyme” (ICE) in mammals. Such ICE and

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related proteins will contribute to apoptosis whereas Ced9 is considered to be homologous of the
proto-oncogene BCl2, which is known as an inhibitor of certain proteases.

Consequently cellular death can be related to a highly regulated process involving both intracellular
and extracellular signals. What is called the “apoptotic cascade” further involves proteins called
caspases which represent powerful effectors of the cellular death. Caspases 8 and 9 for example are
involved in cellular death induced by the membrane receptors Fas and TNF, the mechanism of
which involves increased oxidative stress and ROS. Caspase 3 enzyme is also known to induce
endonucleases activation.

Some of the neurodegenerative diseases have been shown to involve apoptosis. This could be the
case for neuronal degeneration in Alzheimer’s and Parkinson’s diseases but also for ALS and
cerebellar neurodegenerative processes. Such a process, however, is not specific of these
neurological diseases. Apoptosis will also contribute to pigmented retina degeneration or cardiac
stroke. Conversely inhibition of apoptosis is thought to occur in certain cancer such as lymphomas,
lung cancer, ovarian and prostate cancers, etc.

Such general view of apoptosis regulation processes leads to consider the existence of both factors
which contribute to facilitate apoptosis and of factors which inhibit apoptosis.

 Among the factors considered to promote apoptosis it is worth mentioning certain of those
previously mentioned as possible inductors of neurodegenerative diseases such as glutamate
and excitotoxic neurotransmitters, intracellular calcium ions, trophic factors deficit, or
glucocorticoid excessive action. But numerous other factors have been proposed to promote
apoptosis such as neurotoxins, ethanol, ROS and oxidative stress, certain virus, etc.
Oncogenes and tumour suppression gene such as the protein P53 are also considered as
similar factors as well as chemotherapy agents such as cisplatin, doxorubicin or bleomycin.

 Some factors are considered as apoptosis inhibitors, such as trophic factors, oestrogens,
androgens and even some neutral amino acids, certain virus such as adenovirus, baculo-
virus, herpes virus or Epstein-Barr virus, or different pharmacological agents such as calpain
inhibitors, proteases inhibitors, and other tumour promoting agents.

If considering that BCl2 plays a critical role in controlling apoptotic processes it is worth to mention
its putative protective action from cellular death when induced either by irradiation (UV and
gamma), chemotherapy, free radicals, lipid peroxidation, glucose deprivation or even baculo-virus
influence. BCl2, however, is not able to protect against cellular death induced by the Apeptide or
deficits in CNTF trophic factor, which underlines a certain degree of specificity (or limitation) in the
protective action of BCl2.

So, targeting apoptosis could be one of the solutions for the future to limit neuronal death in the
context of the neurodegenerative diseases. Some anti-apoptotic agents have been yet developed
such as propentofyllin, ibedenone or L-threo-dihydroxyphenyl serine. Intracellular signalling
pathways such as PI3K pathways have an inhibitory effect on apoptosis especially through Akt
signalling. But possible tumorigenic effects of these strategies have to be considered.

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 Some other putative protective strategies for neurons in the context of the
neurodegenerative diseases

Besides some direct actions on apoptotic processes and neuronal death mechanisms it could be
proposed some others strategies to promote neuroprotection related to neurodegenerative
diseases. The following procedures could thus be encouraged:

 To limit the action of cytotoxic factors

ROS, oxidative stress, excessive calcium, glutamate and others have been proposed to facilitate
neuronal death in neurodegenerative diseases. This is also the case indirectly for agents contributing
to cardio-vascular diseases and of pro-inflammatory action. In this respect anti-oxidant agents
(vitamin A, C and E, co-enzyme Q, polyphenols, melatonin, etc.) are proposed strategies although we
presently have no evidence for efficacy in clinic. Anti-calcic agents such as type L channels blockers,
calpain inhibitors, or calcineurin activators, are also available compounds but once again with very
few evident results. Similar clinical trials using anti-glutamate compounds including adenosine
receptor agonists to reduce glutamate release were no more effective. However in such category of
compounds riluzole is considered as neuroprotective in ALS and acts at least partly as a glutamate
antagonist. Similarly anti-inflammatory drugs or limiting the metabolism of cholesterol such as the
statins no more show any significant effects although considered as exerting effective protection
against cardio-vascular diseases.

Some other procedures have been proposed such as limiting the effect of NO, stimulating energy
metabolism at cell level (mitochondrial effect) or promoting cytosqueleton stability. But regarding
effective treatment of neurodegenerative diseases the most advanced proposal is related to the
tentative of vaccination against A peptide in an attempt of immunoneurotralization still in a way of
evaluation for Alzheimer’s disease as well as the use of agents putatively destabilizing protein
aggregates (sheet breakers) or inhibiting secretase activity.

 To stimulate endogenous mechanisms potentially neuroprotective in nature but which

could be deficient during the disease progression

Different possibilities have been evidenced:

- To stimulate cAMP and/or cGMP actions;

- To act for stabilizing cellular membrane potential (excitable cells) through stimulation of
inhibitory GABAergic transmission for example;
- To promote endogenous defence mechanisms against oxidative stress and ROS action;
- To promote benefit of oestrogens which may act as putative endogenous neuroprotectors;
- To promote action of trophic factors and especially of neurotrophic factors in case of
neurodegenerative diseases.

Stimulation of cAMP and cGMP pathways will contribute to activate protein kinase A and/or protein
kinase G, the effects of which are among many others to assure optimal functioning of ionic pumps
controlling energetic metabolism of the cell. Such an action is possible using pharmacological
agonists of these nucleotides such as forskolin or antagonists of the phosphodiesterases involved in
their degradation with the view prolonging the effects of cAMP and/or cGMP.

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Stimulation of GABA transmission will use some of the anti-epileptic drugs known in clinic to
reinforce membrane inhibition and acting as GABA receptor agonists. Another possibility is to use
some anaesthetic drugs such as propofol or some agents modulating the GABA A receptors like
barbituric drugs or benzodiazepines.

Antioxidants and oestrogens have been above described as putative agents able to prevent brain
lesion. However, it is possible to fight ROS and oxidative stress by stimulating endogenous protective
mechanisms such as catalase, superoxide dismutase metabolism or glutathion action. Oestrogens
action could be stimulated through administrating some analogs of existing neurosteroids like DHEA,
pregnenolone or 7-hydrosteroids. Indeed, pregnenolone is considered as a metabolic precursor of
neurosteroids synthesis. Clinical results, however, still remains to be evaluated.

Trophic factors have been proposed to represent one of the major issues to prevent or limit brain
lesions. Some of them such as NGF (nerve growth factor), BDNF (brain derived neuronal factor), NT-3
(neurotrophin 3), and NT-4 (neurotrophin 4) or even GDBF (glial cell line derived neuronal factor) are
considered as playing a critical role in the context of the neurodegenerative diseases because of
special relationships with specific neuronal populations involved primarily in some of these diseases.
This is indeed the case for NGF targeting the cholinergic neurons in Alzheimer’s disease, BDNF as a
major factor for dopaminergic neurons survival in Parkinson’s disease, BDNF but also NT-3 and NT-4
for the GABA neurons in Huntington chorea; and BDNF and NT-4 for motor neurons in ALS. The major
difficulty, however, is related to the peptidic nature of these factors and to the absence of
pharmacological agents able to stimulate their brain production of receptors action. Intracerebral
administration of such factors in patients still remains extremely difficult. The idea is therefore to
develop some gene therapy procedure to transfer the gene of the corresponding trophic factor to
the brain of a given patient. Clinical trials have yet been performed for GDNF in Parkinson’s disease,
NGF in Alzheimer’s disease or BDNF in ALS. But data are presently still very limited and rather
disappointing. Because of such difficulties it has been proposed to indirectly stimulate the
production/action of these factors. Gangliosides, lithium or stimulation of excitatory amino acids
receptors have been proposed to be possible candidates for such an indirect action in patients.
Similarly although data remains to be fully evaluated, it has been proposed that physical exercise is a
possible way to stimulate their brain action.

To conclude: neuroprotection in neurodegenerative disease: myth or clinical reality?

Presently clinical results are rather limited or in the way to be evaluated. Two simples remarks can
help to conclude: 1) better understanding of pathophysiological processes in neurodegenerative
disease is the only way to expect developing new therapeutic strategies not aimed at simple
symptomatic effects but also at acting on the pathological processes. So, we have to continue
working to evidence the causes and mechanisms of neuronal death in neurodegenerative diseases;
2) we obviously have to consider that we are in a key period for research because if it is evident that
clinical data are rather disappointing we have also to take into account that in animals models with
all the limitations of these experimental models we are indeed able to modify the brain response to
different sorts of aggression reflecting a huge hope for the future. Because apoptosis is presently
considered as a common final pathway to many degenerative processes we also expect that finding
some ways to reduce apoptosis could be a common way to improve at the same time not only one
but many of the neurodegenerative diseases.