Patent Description:
Neurotrophic factors cerebral dopamine neurotrophic factor (CDNF) and mesencephalic astrocyte-derived neurotrophic factor (MANF) (Lindholm and Saarma, <NUM>; Lindahl et al. , <NUM>) are currently the most efficient proteins for the treatment of rats in the <NUM>-OHDA model of Parkinson's disease (PD). Both factors potently prevent the <NUM>-OHDA-induced behavioral and histological symptoms of Parkinson's disease when applied before the toxin (Lindholm et al. , <NUM>; Voutilainen et al. More importantly, post-treatment (i.e. treatment after <NUM>-OHDA induction) with either factor efficiently restored the normal motor behavior and dopaminergic innervations of the striatum when applied at the stage when the <NUM>-OHDA-induced symptoms of the Parkinson's disease are already far-reaching (Lindholm et al. , <NUM>; Voutilainen et al. CDNF protects and repairs dopamine neurons also in mouse and rhesus monkey MPTP models of Parkinson's disease. In the monkey MPTP model, as well as in the severe rodent <NUM>-OHDA model it is more efficient than glial cell line-derived neurotrophic factor (GDNF) in restoring dopamine neurons in substantia nigra pars compacta (SNPc) and restoring motor behavior (Voutilainen et al. , <NUM>; Airavaara et al. , <NUM>: Voutilainen et al. The mechanisms behind the neuronal protection for these factors are not fully clear but it has been suggested that in addition to the activation of classical survival promoting anti-apoptotic pathways, they regulate unfolded protein response (UPR) pathways, which aim at alleviating oxidative- and ER stress depressing ER-stress-induced apoptotic cell death (Lindahl et al. , <NUM>; Lindahl et al. , <NUM>, Voutilainen et al. Many pathophysiological conditions and degenerative diseases including diabetes mellitus and neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS) and Huntington's disease (HD) are associated with protein misfolding and aggregation that triggers ER stress and activation of the UPR pathways. Accordingly, the effect of CDNF and MANF has been shown in various central nervous system diseases (<CIT>; <CIT>; and Airavaara et al, <NUM>). In addition, CDNF and MANF suppress neuroinflammation, which is involved in the pathophysiology of most if not all CNS diseases and injuries (Nadella et al, <NUM>; Neves et al. , <NUM>; Zhao et al, <NUM>).

Further, <CIT> discloses a genetically-modified non-human animal comprising a disrupted allele for the gene that naturally encodes and expresses a functional MANF gene, wherein said animal displays progressive postnatal reduction of pancreatic beta cell mass due to the disrupted and non-functional MANF gene. A gene therapy vector delivering effective amount of a MANF or CDNF polypeptide or a functional fragment thereof for use in the intrapancreatic treatment of type <NUM> or type <NUM> diabetes is also suggested. Further, Lindahl et al. , <NUM>, disclose that the MANF protein is indispensable for the proliferation and survival of pancreatic beta cells thereby constituting a therapeutic candidate for beta cell protection and regeneration.

<CIT> discloses cell-penetrating MANF or CDNF peptides with the length of <NUM> - <NUM> amino acids comprising the sequence CXXC for use in the treatment of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, stroke, peripheral neuropathy, epilepsy, diabetes or drug addiction.

Structural studies of CDNF and MANF have shown that these proteins consist of two domains: a saposin-like N-terminal domain (Parkash et al. , <NUM>) and a SAP-like C-terminal (Hellman et al. The CXXC motif (residues <NUM>-<NUM> of human MANF, NCBI Reference Sequence: NP_006001. <NUM>) is located in the C-terminal domain (C-MANF) in the loop region outside the helical core of the domain, and the cysteines are connected with the disulfide bond (Hellman et al. Corresponding motif of CDNF is located at the same position (NCBI Reference Sequence: NP_001025125. It has been shown that C-MANF is potently anti-apoptotic in vitro, when expressed inside the sympathetic neurons (Hellman et al. In Lindström et al. , <NUM>, characterization of structural and functional determinants of MANF and CDNF are disclosed.

Cell membranes with their selective permeability control molecular exchanges between the cytosol and the extracellular environment in a similar manner as the intracellular membranes do within the internal compartments. For this reason the plasma membranes often represent a challenging obstacle to the intracellular delivery of many molecules, especially high molecular weight molecules such as full-length proteins. The active transport of high molecular weight molecules through such barrier often requires specific carriers able to cross the lipid bilayer. Cell penetrating peptides (CPPs) are generally <NUM>-<NUM> amino acids long peptides (or motifs within a peptide) which, for their ability to cross cell membranes, are widely used to deliver proteins, plasmid DNA, RNA, oligonucleotides, liposomes and anti-cancer drugs inside the cells (Borrelli et al. , <NUM>; Bode & Löwik, <NUM>; Kalafatovic & Giralt, <NUM>; Kristensen et al.

The present invention is described by the appended claims.

In the present invention, it has been discovered that a C-terminal fragment of the CDNF protein surprisingly protects ER stressed sympathetic and dopaminergic neurons in vitro and in vivo, and in contrast to full-length CDNF the fragment is capable of penetrating neuronal cell membrane as well as the blood-brain-barrier in vivo.

Accordingly, it is an aim of the present invention to provide a C-terminal CDNF fragment with a length of <NUM>-<NUM> amino acids comprising at least the consecutive amino acid residues at positions <NUM>-<NUM> or <NUM>-<NUM> of the sequence as set forth in SEQ ID NO: <NUM>:
<IMG>
or a sequence which has at least <NUM> % sequence identity with the sequence of positions <NUM>-<NUM> or <NUM>-<NUM> in SEQ ID NO: <NUM>, wherein said fragment is a cell membrane penetrating peptide and has a protective effect on neuronal cells, for use as a medicine.

The present invention also provides a pharmaceutical composition comprising said C-terminal CDNF fragment and at least one of the following: physiologically acceptable carrier, buffer, excipient, preservative and stabilizer.

The results of the present invention further provides said C-terminal CDNF fragment for use in the treatment of a degenerative disease or disorder including a central nervous system (CNS) disease, diabetes or a retinal disease, wherein said CNS disease is preferably selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease and other amyloid diseases, multiple system atrophy, amyotrophic lateral sclerosis, frontotemporal lobar degeneration, dementia with Lewy bodies, mild cognitive impairment, traumatic brain injury, peripheral nerve injuries, addiction and stroke.

The aforementioned and other advantages and benefits of the present invention are achieved in the manner described as characteristics in the accompanying claims.

The present disclosure is related to a neurotrophic factor protein CDNF. CDNF polypeptides are the full-length human CDNF with a signal peptide having the total length of <NUM> amino acids and the mature human CDNF without the signal peptide having the total length of <NUM> amino acids (see <FIG>).

Further disclosed but not forming a part of the invention is neurotrophic factor protein MANF. Particularly important MANF polypeptides are the full-length human MANF with a signal peptide having the total length of <NUM> amino acids and the mature human MANF without the signal peptide having the total length of <NUM> amino acids (see <FIG>).

As used herein, the term "C-terminal fragment" as applied to a CDNF or MANF polypeptide, may ordinarily comprise at least about <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM> contiguous or consecutive amino acids, typically, at least about <NUM> contiguous or consecutive amino acids located in the C-terminal SAP-like domain of said polypeptides (See <FIG>). These C-terminal fragments are "functional fragments" retaining at least partly biological activity of the intact polypeptide and may even have properties the intact polypeptide does not have.

In addition to naturally occurring allelic variants of CDNF/MANF, changes can be introduced by mutation into CDNF/MANF nucleic acid sequences that incur alterations such as elongations, insertions and deletions in the amino acid sequences of the encoded CDNF/MANF polypeptide or C-terminal fragment thereof. Nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in the sequence of a CDNF/MANF polypeptide and the C-terminal domain thereof.

A "non-essential" amino acid residue is a residue that can be modified in the wild-type sequences of CDNF/MANF without altering its biological activity, whereas an "essential" amino acid residue is required for such biological activity. For example, amino acid residues that are conserved among the CDNF/MANF molecules of the invention are predicted to be essential and particularly non-amenable to alteration. Amino acids for which conservative substitutions can be made are well known in the art.

Each amino acid can be a natural or non-natural amino acid. The term "non-natural amino acid" refers to an organic compound that is a congener of a natural amino acid in that it has a structure similar to a natural amino acid so that it mimics the structure and reactivity of a natural amino acid. The non-natural amino acid can be a modified amino acid, and/or amino acid analog, that is not one of the <NUM> common naturally occurring amino acids or the rare natural amino acids selenocysteine or pyrolysine. Non-natural amino acids can also be the D-isomer of the natural amino acids. Examples of suitable amino acids include, but are not limited to, alanine, alloisoleucine, arginine, asparagine, aspartic acid, cysteine, cyclohexylalanine, <NUM>,<NUM>-diaminopropionic acid, <NUM>-fluorophenylalanine, glutamine, glutamic acid, glycine, histidine, homoproline, isoleucine, leucine, lysine, methionine, naphthylalanine, norleucine, phenylalanine, phenylglycine, pipecolic acid, proline, pyroglutamic acid, sarcosine, serine, selenocysteine, threonine, tryptophan, tyrosine, valine, a derivative, or combinations thereof.

Certain embodiments of the invention include C-terminal CDNF fragments wherein at least one, two, three, four or more consecutive amino acids have alternating chirality. As used herein, chirality refers to the "D" and "L" isomers of amino acids. In particular embodiments of the invention, at least one, two, three, four or more consecutive amino acids have alternating chirality and the remaining amino acids are L-amino acids.

In present disclosure, the cellular uptake of the C-terminal CDNF fragments of the invention into neuronal cells has been demonstrated. In certain embodiments, uptake is preferably at least <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM> times better compared to full length CDNF or MANF, and with specific peptides even <NUM> times better than full-length CDNF or MANF. In certain embodiments, the invention demonstrates improved cellular uptake efficiency of the C-terminal CDNF fragments of the invention as compared to controls such as full-length human CDNF.

As used herein cellular uptake efficiency refers to the ability of a C-terminal CDNF fragment or C-terminal MANF fragment to traverse a cell membrane. Cellular uptake of the C-terminal CDNF fragments of the invention is not dependent on a receptor or a cell type.

A person skilled in the art can test uptake efficiency of a C-terminal CDNF fragment and/or C-terminal MANF fragment by comparing (i) the amount of a cell-penetrating peptide such as the C-terminal CDNF fragments or C-terminal MANF fragments internalized by a cell type (e.g., neuronal cells, endothelial cells) to (ii) the amount of a control peptide such as full-length CDNF/MANF internalized by the same cell type. To measure cellular uptake efficiency, the cell type may be incubated in the presence of a cell-penetrating peptide such as the C-terminal CDNF fragment or C-terminal MANF fragment for a specified period of time (e.g., <NUM> minutes, <NUM> hour, <NUM> hours, etc.) after which the amount of the cell-penetrating peptide internalized by the cell is quantified. Separately, the same concentration of the control is incubated in the presence of the cell type over the same period of time, and the amount of the second peptide internalized by the cell is quantified. Quantification can be achieved by fluorescently labeling the cell-penetrating peptide such as the C-terminal CDNF fragments or C-terminal MANF fragments (e.g., with a FITC dye) and measuring the fluorescence intensity using techniques well-known in the art.

The C-terminal CDNF fragments of the invention also demonstrate protective effect for cells, e.g. neuronal cells as compared to suitable controls (see e.g. <FIG> and <FIG>). As used herein protective effect refers to the ability of a C-terminal CDNF fragments of the invention to promote survival of, e.g., dopaminergic neurons or ER stressed neuronal cells. A person skilled in the art can test said protective effect by comparing (i) the dose of a C-terminal CDNF fragments of the invention to survival of a cell type (e.g., sympathetic neuronal cells or dopaminergic neurons) to (ii) the level of survival of control peptide by the same cell type or to the level of survival of no added neurotrophic factors by the same cell type. To measure cell survival, the cell type may be incubated in the presence of a C-terminal CDNF fragments of the invention for a specified period of time (e.g., <NUM> minutes, <NUM> hour, <NUM> hours, etc.) after which the cell survival of the cell is quantified. Separately, the same concentration of control peptide is incubated in the presence of the cell type over the same period of time, and the cell survival of the second peptide by the cell is quantified. Alternatively, the cell type is incubated without neurotrophic factors over the same period of time, and the cell survival by the cell is quantified.

In an embodiment, to measure cell survival, the cell type may be injected with a C-terminal CDNF fragments of the invention and incubated for a specified period of time (e.g., <NUM> minutes, <NUM> hour, <NUM> hours, etc.) after which the cell survival of the cell is quantified. Control cells are injected with a buffer (i.e. with no neurotrophic factors) and the control cells are incubated over the same period of time, and the cell survival by the cell is quantified.

In certain embodiments, protective effect (measured as cell survival) of the C-terminal CDNF fragment of the invention is at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least1. <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM> -fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, at least <NUM>-fold, or at least <NUM>-fold compared to the cells incubated in presence of no added growth factors or injected with a buffer without growth factors.

In an embodiment, the protective effect is at least <NUM>-fold compared to the cells incubated without growth factors.

In an embodiment, the protective effect is at least1. <NUM>-fold compared to the cells incubated without growth factors.

Accordingly, the present disclosure provides a C-terminal CDNF fragment with a length of <NUM>-<NUM> amino acids comprising at least the consecutive amino acid residues at positions <NUM>-<NUM> or <NUM>-<NUM> of the sequence as set forth in SEQ ID NO: <NUM>:
<IMG>
or a sequence which has at least <NUM> % homology or sequence identity with the sequence of positions <NUM>-<NUM> or <NUM>-<NUM> in SEQ ID NO:<NUM>, wherein said fragment is a cell membrane penetrating peptide and has a protective effect on neuronal cells, preferably for use as a medicine. In an embodiment, the C-terminal CDNF fragment comprises or consist of a sequence which has at least <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM>%, or <NUM>% homology or sequence identity with the sequence of positions <NUM>-<NUM> or <NUM>-<NUM> in SEQ ID NO:<NUM>.

In a preferred embodiment, the fragment, preferably with a length of <NUM>-<NUM> amino acids, comprises at least the consecutive amino acid residues at positions <NUM>-<NUM> or <NUM>-<NUM> of the sequence as set forth in SEQ ID NO:<NUM> or a sequence which has at least <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM>%, or <NUM>% homology or sequence identity with the sequence of positions <NUM>-<NUM> or <NUM>-<NUM> in SEQ ID NO:<NUM>, and, if present, the sequence flanking said consecutive amino acid residues preferably has at least <NUM> % homology or sequence identity with the sequence at corresponding positions in SEQ ID NO:<NUM>. The term "flanking sequence" refers to amino acids elongating both or at least one of the terminal ends of said consecutive amino acid residues. In an embodiment, said sequence flanking said consecutive amino acid residues preferably has at least <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM>%, or <NUM>% homology or sequence identity with the sequence at corresponding positions in SEQ ID NO:<NUM>.

Without wishing to be limited by any theory, the results of <FIG> show that the amino acid sequence motifs important for cell membrane penetration and for protective effect on neuronal cells are located between the amino acid residues at positions about <NUM> and about <NUM> of SEQ ID NO:<NUM>. This area contains two helix structures (helix <NUM> and helix <NUM>) and the CXXC motif in-between (see <FIG>). Further, the results of <FIG> and <FIG> show that C-CDNF peptide <NUM> (<NUM> aa) containing helices <NUM> and <NUM> and the CXXC motif has protective effect on neuronal cells. Accordingly, the present invention is directed to embodiments, wherein the C-terminal CDNF fragment comprises at least the consecutive amino acid residues at positions <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM> or <NUM>-<NUM> of the sequence as set forth in SEQ ID NO:<NUM> or a sequence which has at least <NUM> % homology or sequence identity with the sequence of positions <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM> or <NUM>-<NUM> in SEQ ID NO:<NUM>, and, if present, the sequence flanking said consecutive amino acid residues preferably has at least <NUM> % homology or sequence identity with the sequence at corresponding positions in SEQ ID NO:<NUM>. In an embodiment, the C-terminal CDNF fragment comprises or consist of a sequence which has at least <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM>%, or <NUM>% homology or sequence identity with the sequence of positions <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM> or <NUM>-<NUM> in SEQ ID NO:<NUM>. In an embodiment, the sequence flanking said consecutive amino acid residues, if present, preferably has at least <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM>%, or <NUM>% homology or sequence identity with the sequence at corresponding positions in SEQ ID NO:<NUM>.

In another preferred embodiment, the C-terminal CDNF fragment comprises or consists of at least the consecutive amino acid residues at positions <NUM>-<NUM> (peptide <NUM>; SEQ ID NO: <NUM>), <NUM>-<NUM> (peptide <NUM>; SEQ ID NO: <NUM>), <NUM>-<NUM> (peptide <NUM>; SEQ ID NO: <NUM>), <NUM>-<NUM> (peptide <NUM>; SEQ ID NO: <NUM>), or <NUM>-<NUM> (peptide <NUM>; SEQ ID NO: <NUM>) of the sequence as set forth in SEQ ID NO:<NUM> or a sequence which has at least <NUM> % homology or sequence identity with the sequence of positions <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, or <NUM>-<NUM> in SEQ ID NO:<NUM>, and, if present, the sequence flanking said consecutive amino acid residues preferably has at least <NUM> % homology or sequence identity with the sequence at corresponding positions in SEQ ID NO:<NUM>. In an embodiment, the C-terminal CDNF fragment comprises or consist of a sequence which has at least <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM>%, or <NUM>% homology or sequence identity with the sequence of positions <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, or <NUM>-<NUM> in SEQ ID NO:<NUM>. In an embodiment, the sequence flanking said consecutive amino acid residues preferably has at least <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM>%, or <NUM>% homology or sequence identity with the sequence at corresponding positions in SEQ ID NO:<NUM>.

In another preferred embodiment, the C-terminal CDNF fragment comprises or consists of at least the consecutive amino acid residues at positions <NUM>-<NUM> of the sequence as set forth in SEQ ID NO:<NUM> or a sequence which has at least <NUM> % homology or sequence identity with the sequence of positions <NUM>-<NUM> in SEQ ID NO:<NUM> and, if present, the sequence flanking said consecutive amino acid residues preferably has at least <NUM> % homology or sequence identity with the sequence at corresponding positions in SEQ ID NO:<NUM>. In an embodiment, the C-terminal CDNF fragment comprises or consist of a sequence which has at least <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM>%, or <NUM>% homology or sequence identity with the sequence of positions <NUM>-<NUM> in SEQ ID NO:<NUM>.

As used herein in the specification and in the claims section below, the term "fragment" includes native peptides (either degradation products, synthetically synthesized peptides or recombinant peptides) and modified peptides, which may have, for example, modifications rendering the peptides more stable or less immunogenic. Such modifications include, but are not limited to, cyclization, N-terminus modification, C-terminus modification, peptide bond modification, backbone modification and residue modification. The fragment may also comprise further elongations, deletions, substitutions or insertions.

In an embodiment, the fragment is resistant to protease cleavage. In an embodiment, the fragment comprises an elongation, deletion, insertion, substitution or modification such that said elongation, deletion, insertion, substitution or modification abolishes at least one protease cleavage site.

As used herein, "protease cleavage site" refers to an amino acid sequence that is recognized and cleaved by a protease. In some embodiments, C-terminal CDNF fragment includes one or more protease cleavage sites that can be cleaved by a cysteine protease, a metalloprotease, or a serine protease. In some embodiments, the protease cleavage sites are the protease cleavage sites as as illustrated, for example, in <FIG> or in <FIG> or in Tables <NUM> and <NUM>.

As used herein, the term "protease-resistant fragment" or "fragment is resistant to protease cleavage" refers to a C-terminal CDNF fragment or C-terminal MANF fragment containing altered amino acid sequence that abolishes at least one native protease cleavage site or changes a sequence close or adjacent to a native protease cleavage site such that the protease cleavage is prevented, inhibited, reduced, or slowed down as compared to corresponding native C-terminal CDNF fragment or C-terminal MANF fragment.

In some embodiments, a suitable alteration is abolishes at least one protease cleavage site.

In some embodiments, a suitable alteration abolishes at least two protease cleavage sites.

In some embodiments, a suitable alteration abolishes at least three protease cleavage sites.

In some embodiments, a suitable alteration abolishes at least four or more protease cleavage sites.

In an embodiment, all cysteine protease cleavage sites have been abolished.

In an embodiment, all metalloprotease cleavage sites have been abolished.

In an embodiment, all serine protease cleavage sites have been abolished.

An alteration can be amino acid substitutions, deletions, insertions, elongations or modifications.

For example, any one amino acid within the region corresponding to residues <NUM>-<NUM> (e.g., <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM> and <NUM>-<NUM>) of SEQ ID NO:<NUM>, or corresponding amino acids in SEQ ID NOs: <NUM>, <NUM>, and <NUM>-<NUM>, can be substituted with any other amino acid, deleted or modified. For example, substitutions at positions adjacent to a protease cleavage site may affect protease recognition of the cleavage site. Substitution or insertion of one or more additional amino acids within each recognition site may abolish one or more protease cleavage sites. Deletion of one or more of the residues in the degenerate positions may also abolish both protease cleavage sites.

In some embodiments, a protease-resistant fragment contains amino acid substitutions or modifications at positions corresponding to <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> of SEQ ID NO:<NUM>, or corresponding amino acids in SEQ ID NOs: <NUM>, <NUM>, and <NUM>-<NUM>.

In some embodiments, the protease-resistant fragment suitable for the invention may contain additional alterations. For example, up to <NUM>% or more of the residues of SEQ ID NO:<NUM>, NO:<NUM>, NO:<NUM> or NO: <NUM>-<NUM> may be changed (e.g., up to <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, or more residues may be changed or altered). Thus, a protease-resistant fragment suitable for the invention may have an amino acid sequence at least <NUM>%, including at least <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>%, identical to SEQ ID NO:<NUM>, NO:<NUM>, NO:<NUM>, or NO:<NUM>-<NUM>.

As used herein, the term "cysteine protease protease cleavage site" (also referred to as "cysteine protease cleavage site" or "cysteine protease cleavage sequence") refers to the amino acid sequence of a peptide or protein that serves as a recognition sequence for enzymatic protease cleavage by cysteine protease. In some embodiments, a cysteine cleavage site may comprise QILH|SWGE or HSWG|EECR in SEQ ID NO:<NUM>, or corresponding amino acids in SEQ ID NOs: <NUM>, <NUM>, and <NUM>-<NUM>, wherein the cleavage site is shown as "|" in the sequence.

As used herein, the term "metalloprotease cleavage site" (also referred to as "metalloprotease cleavage site" or "metalloprotease cleavage sequence") refers to the amino acid sequence of a peptide or protein that serves as a recognition sequence for enzymatic protease cleavage by metalloprotease. In some embodiments, a metalloprotease cleavage site may comprise DLRK|MRVA, DYVN|LIQE, MPAM|KICE, QICE|LKYE, LAPK|YAAT, EKTD|YVNL or RVAE|LKQI in SEQ ID NO:<NUM>, or corresponding amino acids in SEQ ID NOs: <NUM>, <NUM>, and <NUM>-<NUM>, wherein the cleavage site is shown as "|" in the sequence.

As used herein, the term "serine protease cleavage site" (also referred to as "serine protease cleavage site" or "serine protease cleavage sequence") refers to the amino acid sequence of a peptide or protein that serves as a recognition sequence for enzymatic protease cleavage by serine protease. In some embodiments, a serine cleavage site may comprise VAEL|KQIL, LDLA|SVDL or TDYV|NLIQ in SEQ ID NO:<NUM>, or corresponding amino acids in SEQ ID NOs: <NUM>, <NUM>, and <NUM>-<NUM>, wherein the cleavage site is shown as "|" in the sequence.

In an embodiment, the protease is selected from the group consisting of a cysteine protease, a metalloprotease, and a serine protease.

In an embodiment, the cysteine protease is cathepsin K.

In an embodiment, the metalloprotease is MMP-<NUM> or MMP-<NUM>.

In an embodiment, the serine protease is chymotrypsin A or elastase-<NUM>.

In an embodiment, the C-terminal CDNF fragment consists of <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or 53amino acids. The fragments may comprise any of the naturally occurring amino acids such as alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine as well as non-conventional or modified amino acids. Preferably, the fragment has at least <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, or <NUM>% homology or sequence identity with the sequence of the C-terminal domain in the human CDNF protein. More preferably, the fragment has at least <NUM>% homology or sequence identity with the sequence of the C-terminal domain in the human CDNF protein. "Homology" as used herein refers to sequence similarity between a reference sequence and at least a fragment of a second sequence. As described below, BLAST will compare sequences based upon percent identity and similarity.

The terms "identical" or percent "identity," in the context of two or more amino acid sequences, refers to two or more sequences or subsequences that are the same. Two sequences are "substantially identical" if two sequences have a specified percentage of amino acid residues that are the same (i.e., <NUM>% identity, optionally <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% or <NUM>% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Optionally, the identity exists over a region that is at least about <NUM> amino acids in length, or more preferably over a region that is <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or more amino acids in length.

For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. When comparing two sequences for identity, it is not necessary that the sequences be contiguous, but any gap would carry with it a penalty that would reduce the overall percent identity.

A "comparison window," as used herein, includes reference to a segment of any one of the number of contiguous positions in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well known in the art such as ClustalW or FASTA.

Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST <NUM> algorithms, which are described in <NPL> and <NPL>, respectively. For amino acid sequences, the BLASTP program uses as defaults a wordlength of <NUM>, and expectation (E) of <NUM>, and the BLOSUM62 scoring matrix [see <NPL>] alignments (B) of <NUM>, expectation (E) of <NUM>, M=<NUM>, N=-<NUM>, and a comparison of both strands. For short amino acid sequences, PAM30 scoring matrix can be applied.

The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., <NPL>). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two amino acid sequences would occur by chance.

Preferably, the C-terminal CDNF which is at least <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM>%, or <NUM>% homologous to the sequence of SEQ ID NO:<NUM> comprises sequence CXXC in positions <NUM>-<NUM> of SEQ ID NO:<NUM>, wherein X is any amino acid.

In another preferred embodiment, said sequence which is at least <NUM> %,<NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM> %, <NUM>%, or <NUM>% homologous to the sequence of SEQ ID NO:<NUM> comprises sequence CKGC in positions <NUM>-<NUM> of SEQ ID NO:<NUM>.

In an embodiment, the C-terminal CDNF fragment does not contain its natural C-terminal amino acids, i.e. the ER retention signal. Accordingly, in a preferred embodiment the fragment lacks the ER retention signal KTEL corresponding to positions <NUM>-<NUM> of SEQ ID NO:<NUM>.

The present invention also shows that the fragment may be conjugated to a detectable chemical or biochemical moiety such as a FITC-label. As used herein, a "detectable chemical or biochemical moiety" means a tag that exhibits an amino acid sequence or a detectable chemical or biochemical moiety for the purpose of facilitating detection of the peptide; such as a detectable molecule selected from among: a visible, fluorescent, chemiluminescent, or other detectable dye; an enzyme that is detectable in the presence of a substrate, e.g., an alkaline phosphatase with NBT plus BCIP or a peroxidase with a suitable substrate; a detectable protein, e.g., a green fluorescent protein. Preferably, the tag does not prevent or hinder the penetration of the fragment into a target cell.

N- and/or C-terminal modifications of the C-terminal CDNF fragments to increase the stability and/or cell permeability of the fragments are also preferred. Acetylation - amidation of the termini of the CDNF fragment (i.e. N-terminal acetylation and C-terminal amidation) is one of the options known in the art (see, e.g., <NPL>).

Since both the C-terminal CDNF fragment and the C-terminal MANF fragment potently protected the dopamine neurons from death (see <FIG> and <FIG>), the prior art such as <CIT>, and <CIT> shows that the fragments can be used in the treatment of central nervous system (CNS) diseases such as Alzheimer's disease, Parkinson's disease (PD), multiple system atrophy, amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration, dementia with Lewy bodies, mild cognitive impairment, Huntington's disease (HD), traumatic brain injury, drug addiction and stroke. Further results supporting the present invention are provided in <FIG> showing the effect of C-CDNF in a PD model and in <FIG> and <FIG> showing the effect of C-CDNF in an ALS model. It is also notable that a short MANF peptide (MANF4) is not effective in the rat <NUM>-OHDA model of Parkinson's disease when tested in neurorestorative, more clinically oriented set up i.e. when added after <NUM>-OHDA (see <FIG>).

The effects of C-terminal CDNF fragment or the C-terminal MANF fragment in CNS include targeting neurons but also other cell types in the CNS such as microglia, astrocytes and neural stem cells or a neuronal precursor cell, and besides survival also any other property they have such as migration, proliferation, differentiation and maturation.

Our results shown in <FIG> confirm that the C-terminal MANF fragment is effective in the treatment of type I and type II diabetes. Further, <CIT> discloses that CDNF and MANF are also active in retinal disorders. Accordingly, the present invention is directed to a treatment of said central nervous system (CNS) diseases, diabetes and retinal disorders. ER-stress-induced apoptotic cell death also contribute to other degenerative diseases in which the function or structure of the affected tissues or organs will progressively deteriorate over time (for a review, see <NPL>). Some further examples of such degenerative diseases are age-related macular degeneration, Stargardt disease, glaucoma, retinitis pigmentosa, and optic nerve degeneration; Niemann-Pick disease; atherosclerosis; progressive supranuclear palsy; cancer; Tay-Sachs disease; keratoconus; inflammatory bowel disease (IBD); prostatitis; osteoarthritis; osteoporosis; and rheumatoid arthritis as well as more acute conditions such as a traumatic brain injury or an ischemia-reperfusion injury, e.g., myocardial ischemic injury, renal ischemic injury, or stroke. The present invention is thus also directed to a treatment of a degenerative disease or disorder.

In a method of treatment, a pharmaceutically effective amount of the C-terminal fragment is administered to a patient. In other words, the fragment according to the present invention is for use in the treatment of a degenerative disease or disorder including central nervous system (CNS) diseases and other neurological disorders such as Alzheimer's disease, Parkinson's disease (PD), non-motor symptoms of PD (such as constipation, depression and hallucinations), multiple system atrophy, amyotrophic lateral sclerosis, ischemic stroke, peripheral neuropathy, frontotemporal lobar degeneration, dementia with Lewy bodies, mild cognitive impairment, Huntington's disease, epilepsy, traumatic brain injury, peripheral nerve injuries, hemorrhagic stroke or addiction (e.g., abuse of cocaine, morphine, amphetamine, or alcohol), and type I and type II diabetes or retinal disorders. More preferably, the fragment is for use in the treatment of Parkinson's disease or amyotrophic lateral sclerosis.

The actual dosage amount of the C-terminal fragment of CDNF or MANF (e.g., an effective amount) that is administered to a patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. The practitioner responsible for administration can determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

In one embodiment of the present invention, the C-terminal CDNF fragment can be incorporated into pharmaceutical compositions. Such compositions of the invention are prepared for storage by mixing the peptide having the desired degree of purity with optional physiologically acceptable carriers (such as nanocarriers), excipients, or stabilizers (<NPL>)), in the form of lyophilized cake or aqueous solutions. Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about <NUM> residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counter-ions such as sodium; and/or non-ionic surfactants such as Tween, Pluronics or polyethylene glycol (PEG).

The fragment may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively), in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, supra.

In an embodiment, pharmaceutical compositions may comprise, for example, at least about <NUM>% of an active compound. In other embodiments, an active compound may comprise between about <NUM>% to about <NUM>% of the weight of the unit, or between about <NUM>% to about <NUM>%, for example, and any range derivable therein.

In other non-limiting examples, a dose of a pharmaceutical composition or formulation can comprise from about <NUM> ng/kg/body weight of C-terminal CDNF fragment, about <NUM> ng/kg/body weight, about <NUM> ng/kg/body weight, about <NUM> ng/kg/body weight, about <NUM> ng/kg/body weight, about <NUM> ng/kg/body weight, about <NUM> ng/kg/body weight, about <NUM> ng/kg/body weight, <NUM>µg/kg/body weight, about <NUM>µg/kg/body weight, about <NUM>µg/kg/body weight, about <NUM>µg/kg/body weight, about <NUM>µg/kg/body weight, about <NUM>µg/kg/body weight, about <NUM>µg/kg/body weight, about <NUM>µg/kg/body weight, about <NUM> milligram/kg/body weight, about <NUM> milligram/kg/body weight, about <NUM> milligram/kg/body weight, about <NUM> milligram/kg/body weight, about <NUM> milligram/kg/body weight, about <NUM> milligram/kg/body weight, about <NUM> milligram/kg/body weight, about <NUM> milligram/kg/body weight, to about <NUM>/kg/body weight of C-terminal CDNF fragment or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about <NUM>/kg/body weight to about <NUM>/kg/body weight, about <NUM>µg/kg/body weight to about <NUM> milligram/kg/body weight of C-terminal CDNF fragment, etc., can be administered, based on the numbers described above.

The invention also features a pharmaceutical composition that can further include a neural cell. The neural cell can be, for example, a neuron, a neural stem cell, or a neuronal precursor cell.

In another embodiment, the pharmaceutical composition comprises a therapeutically effective amount of recombinant vectors comprising a nucleotide sequence that encodes a C-terminal fragment as defined above, recombinant viral vectors comprising a nucleotide sequence that encodes a C-terminal fragment as defined above, or a host cell expressing a C-terminal fragment as defined above. Said viral vector is preferably selected from the group consisting of an adenovirus, an adeno-associated virus, a retrovirus such as a lentivirus, herpes virus, and papillomavirus comprising a polynucleotide encoding a C-terminal fragment as defined above. Typically the recombinant vectors and recombinant viral vectors include expression control sequences like tissue- or cell-type specific promoters that direct the expression of the polynucleotide of the invention in various systems, both in vitro and in vivo. Vectors can also be hybrid vectors that contain regulatory elements necessary for expression in more than one system. Vectors containing these various regulatory systems are commercially available and one skilled in the art will readily be able to clone the C-terminal fragment as defined herein into such vectors. Selection of recombinant viral vectors suitable for use in the invention, methods for inserting nucleic acid sequences for expressing the C-terminal fragment into the vector, and methods of delivering the viral vector to the cells of interest are within the skill in the art. See, for example, <NPL>.

The route of administration is in accord with known methods as well as the general routes of injection or infusion by intravenous or peripheral administration, intraperitoneal, subcutaneous, intrathecal, intracerebroventricular, intranasal, transdermal, intracerebral, intramuscular, intraocular, intraarterial, or intralesional means, or sustained release systems as noted below. The C-terminal fragment or a pharmaceutical composition comprising said fragment can be administered continuously by infusion or by bolus injection. Generally, where the disorder permits, one should formulate and dose the fragment for site-specific delivery. Administration can be continuous or periodic. Administration can be accomplished by a constant- or programmable-flow implantable pump or by periodic injections. Peripheral or systemic administration is preferred as the present invention shows that both C-terminal MANF and CDNF fragments are capable of penetrating neuronal cell membrane as well as the blood-brain-barrier (see <FIG> and <FIG>). Other preferred administration routes are subcutaneous, intrathecal, intracerebroventricular, intranasal, or transdermal administration. In <FIG>, the effect of a subcutaneous injection of C-CDNF protein is shown in rats having an induced cerebral stroke.

Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the fragment, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels as described by <NPL>) and <NPL>) or polyvinylalcohol, polylactides (<CIT>, <CIT>), or non-degradable ethylene-vinyl acetate (Langer et al.

Gene therapy vectors can be delivered to a subject using corresponding administration modes as defined above for the peptide fragment, preferably by, for example, intravenous injection, or by intraperitoneal, subcutaneous, intrathecal, or intracerebroventricular administration. The pharmaceutical preparation of a gene therapy vector can include an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is embedded.

The sequence alignment of <FIG> shows the high sequence identity of C-terminal CDNF and MANF peptides. Accordingly, the present inventors extrapolate from the results presented herein that also in the C-terminal MANF fragment the amino acid sequence motifs important for cell membrane penetration and for protective effect on neuronal cells are located between the amino acid residues at positions about <NUM> and about <NUM> of SEQ ID NO:<NUM> corresponding to positions <NUM>-<NUM> in SEQ ID NO:<NUM> and about <NUM> and <NUM> of SEQ ID NO:<NUM> corresponding to positions <NUM>-<NUM> in SEQ ID NO:<NUM>.

The invention further provides an expression vector encoding said isolated polynucleotide and a host cell transformed with said vector. The selection of recombinant vectors suitable for expressing said isolated polynucleotide, methods for inserting nucleic acid sequences for expressing the C-CDNF fragment into the vector, and methods of delivering the recombinant vector to the cells of interest are within the skill in the art. See, for example <NPL>.

The publications and other materials used herein to illuminate the background of the invention, and in particular, to provide additional details with respect to its practice, are incorporated herein by reference. The present invention is further described in the following examples, which are not intended to limit the scope of the invention.

For the cultures of sympathetic neurons (Hellman et al. , <NUM>; Hamner et al. , <NUM>; Lindholm et al. , <NUM>; Sun et al. , <NUM>; Aalto et al. , <NUM>) the superior cervical ganglia from the postnatal day (P) <NUM>-<NUM> mice were digested with collagenase (<NUM>/ml; Worthington), dispase (<NUM>/ml; Roche Molecular Biochemicals), and trypsin (<NUM>/ml; Worthington) for <NUM> at <NUM> and dissociated mechanically with a siliconized glass Pasteur pipette. Non-neuronal cells were removed by extensive preplating. Almost pure neurons were cultured in polyornithine/laminin (Sigma)-coated <NUM>-mm plastic dishes in the small-size standard microislands in the Neurobasal medium and B27 supplement (Invitrogen/Gibco) in the presence of <NUM> ng/ml mouse nerve growth factor (NGF) (Promega) for <NUM>-<NUM> days. NGF was deprived by extensive washing and addition of the function-blocking anti-NGF antibodies (Roche). The neurons were pressure-microinjected with special neuronal microinjection equipment (Hellman et al. , <NUM>; Hamner et al. , <NUM>; Lindholm et al. , <NUM>; Sun et al. , <NUM>; Yu et al. , <NUM>)(Sun et al. , <NUM>; Sun et al. For the survival assay, all neurons on the microislands were counted in the beginning (initial number) and the end (three days) of the experiment and expressed as % of initial.

Microinjection of the sympathetic neurons was performed as described earlier (<NPL>). Plasmids for CDNF have been described earlier. Briefly, newborn mouse SCG neurons were grown with NGF (Promega) for <NUM>-<NUM> days then the nuclei were microinjected with the expression plasmids for full-length (FL)-CDNF and C-CDNF together with a reporter plasmid for enhanced green fluorescent protein (EGFP), using vector concentration of <NUM> ng/ul in each experiment. Similar results were achieved with plasmid concentrations of <NUM> ng/ul. For protein microinjection, recombinant full length (FL)-CDNF, C-CDNF proteins in PBS at 200ng/ul were microinjected directly into the cytoplasm together with fluorescent reporter Dextran Texas Red (MW <NUM> Da) (Invitrogen, Molecular Probes) that facilitates identification of the successfully injected neurons. Next day tunicamycin (<NUM>) was added, and after <NUM> days the living fluorescent neurons were counted. Living fluorescent (EGFP-expressing or Dextran Texas Red-containing) neurons were "blindly" counted three days later and expressed as percent of initial living fluorescent neurons counted <NUM>-<NUM> hours after microinjection. The experiments with plasmids were repeated on independent cultures <NUM> times for plasmid experiment, whereas four independent protein injection experiments were performed. On average, <NUM>-<NUM> neurons were successfully injected per experimental group. The results were expressed as the mean ± the SEM. Data of each experimental group was compared with control plasmid PCR3. <NUM> (vector) or PBS (in the protein injection experiments) by one-way ANOVA and post hoc Dunnett's t test. The null hypothesis was rejected at p < <NUM>.

Constructs coding for full-length (FL) or carboxy-terminal (C) domains were inserted to pCR3. <NUM> vector (Invitrogen) either by TOPO/TA cloning system (Invitrogen) or by using restriction endonucleases. Full-length CDNF in pCR3. <NUM> vector are <NUM> bp (<NUM> amino acids) and 561bp (<NUM> amino acids) amino acids long, respectively and have in their N-termini signal sequence for ER targeting. C-CDNF is 186bp long, corresponding to amino acids <NUM>-<NUM> in FL-CDNF.

Human recombinant CDNF (full length pre-CDNF consisting of the <NUM> amino acids, with <NUM> amino acids long signal sequence and <NUM> amino acids long mature CDNF sequence), human N-CDNF (consisting of human CDNF signal sequence of <NUM> amino acids and the part of the mature CDNF from amino acid <NUM>- amino acid <NUM>) and human C-CDNF (consisting of <NUM> amino acids long CDNF signal sequence fused with the C-terminal domain of mature CDNF starting from amino acid <NUM> spanning to amino acid <NUM>).

Human recombinant MANF (full length pre-MANF consisting of the <NUM> amino acids, with <NUM> amino acids long signal sequence and <NUM> amino acids long mature MANF sequence), human N-MANF (consisting of human MANF signal sequence of <NUM> amino acids and the part of the mature MANF from amino acid <NUM>- amino acid <NUM>) and human C-MANF (consisting of <NUM> amino acids long CDNF signal sequence fused with the C-terminal domain of mature MANF starting from amino acid <NUM> spanning to amino acid <NUM>).

Codon optimized cDNA synthesis for hMANF and hCDNF and their domains was ordered from Genewiz and respective pQMCF expression vectors were constructed. N-CDNF, C-CDNF, N-MANF and C-MANF constructs had Histidine tag at the C-terminus. cDNA-s were verified by sequencing in final vectors. hMANF and hCDNF proteins were produced by CHO-derived suspension cell line CHOEBNALT85, chemically defined serum-free media was used for culturing of the cells.

CHOEBNALT85 cells were transfected with <NUM>µg of the expression plasmids. <NUM> after the transfection <NUM>µg/ml of G418 was added to select plasmid containing cell population. The expression and secretion of the proteins were analyzed <NUM> after transfection in reduced conditions in cell lysates and in supernatants.

hMANF and hCDNF proteins were purified by two-step ion-exchange chromatography and gelfiltrated into PBS, pH <NUM>. Based on the SDS-PAGE and Western blotting analysis with CDNF and MANF antibodies (MANF 4E12-HRP and CDNF-7D6-HRP, Icosagen Tartu, Estonia) the used proteins were more than <NUM>% pure.

CDNF and MANF domains were purified on Ni-affinity column and the proteins were also analysed buy SDS-PAGE and Western blotting using mouse monoclonal antibody to His tag (Cat No. A00186; GeneScript).

The produced proteins had the following sequence:.

To study the dopamine neurons (Yu et al. , <NUM>; Yu and Arumae, <NUM>), the midbrain floors were dissected from the ventral mesencephali of <NUM>-d-old NMRI strain mouse embryos. Tissues were incubated with <NUM>% trypsin (ICN Biomedical), then mechanically dissociated using a large fire-polished Pasteur pipette. The neurons were grown on the poly-L-ornithine-coated (Sigma) <NUM>-well culture plates in DMEM/F12 medium (Invitrogen) containing N2 supplement (Invitrogen) in the presence or absence of GDNF (<NUM> ng/ml) or with CDNF, MANF, C-CDNF and C-MANF polypeptides at different concentrations for five days. Same amount of the neurons were plated to each well at the beginning of the experiments. The cultures without added neurotrophic factors served as the negative control. As the midbrain cultures contain several neuronal types, the cultures were fixed and immunostained with the antibodies to tyrosine hydroxylase (TH) (Millipore), a specific marker for the dopaminergic neurons. Images of each well were scanned by CellInsightTM and immunopositive neurons were counted by CellProfiler and CellProfiler analyst software. Data are expressed as a percentage of GDNF-maintained TH-positive neurons. All experiments were repeated at least three times on the independent cultures. The results were expressed as the mean the SEM and were tested for the significance by either one-way ANOVA and Tukey's post hoc test or by two-tailed Student's t-test. The null hypothesis was rejected at P ≤<NUM>.

CDNF, C-CDNF and C-MANF were iodinated with <NUM>I-Na using the lactoperoxidase method. The protein in question was dissolved in <NUM>µl of <NUM> phosphate buffer, pH <NUM>, and mixed with <NUM>I-Na (<NUM> mCi /<NUM>µl; <NUM> mCi= <NUM> mBq; GE Healthcare). The reaction was started by adding lactoperoxidase <NUM>µl of 50µg/ml and <NUM>% H<NUM>O<NUM>. The mixture was incubated at room temperature for <NUM> and the reaction was stopped by adding <NUM> volumes of <NUM> phosphate buffer, pH <NUM>, containing <NUM> NaI, <NUM> NaCl, and then 25µl of <NUM>% BSA was added. Free iodine and iodinated protein were separated by gel filtration on Sephadex G-<NUM> columns (PM10; GE Healthcare). For column equilibrium and elution, <NUM> phosphate buffer, pH <NUM>, with <NUM>% BSA was used. The iodinated growth factors were sometimes concentrated by using YM-<NUM> Centricon columns (Millipore). The specific activity of <NUM>I-labeled CDNF, C-CDNF and C-MANF was measured on the Wizard <NUM><NUM> Automatic Gamma Counter (Perkin Elmer, Wallac) and was about <NUM><NUM> cpm/µg protein. The labelled protein were kept at <NUM> and used within <NUM> weeks after labelling.

<NUM> dopamine neurons grown on the <NUM> well plate in culture were incubated with <NUM>,<NUM> cpm of iodinated CDNF or C-CDNF per well at <NUM> <NUM> hr. The cells were transferred to ice and washed once with <NUM> of the ice cold media. Then cells were transferred to Eppendorf tubes and washed at <NUM> once with <NUM> acetic acid, <NUM> NaCl, pH <NUM>. After centrifugation at <NUM> for <NUM> cells were dissolved in <NUM> of <NUM> N NaOH and counted on the gamma counter.

<NUM>I- CDNF, <NUM>I C-CDNF or <NUM>I C-MANF (all proteins at <NUM><NUM> cpm in <NUM>µl) were injected subcutaneously to adult male Wistar rats. Animals were perfused with PBS <NUM> hours later. Radioactivity was analyzed in different brain regions by gamma counter. Data are shown as mean ±SEM. The differences between groups were analyzed ANOVA followed by Tukey-Kramers post hoc test.

<NUM> pheochromocytoma cells were grown on the <NUM> well plate in DMEM culture with <NUM>% FCS and <NUM>% horse serum. The cells were washed with PBS and were incubated with <NUM>,<NUM> cpm of iodinated CDNF or C-CDNF per well at <NUM> <NUM>. The cells were put on ice, washed once with <NUM> of the ice cold media. Then cells were transferred to Eppendorf tubes and washed once with <NUM> acetic acid, <NUM> NaCl, pH <NUM>. After centrifugation at <NUM> for <NUM> cells were dissolved in <NUM> of <NUM> N NaOH and counted in Wallac gamma counter.

In neurorestoration model of PD, rats were lesioned with <NUM>-OHDA as described before (Voutilainen et al. , <NUM>; Voutilainen et al. , <NUM>, Penttinen et al. Briefly, rats received under isoflurane anesthesia unilateral stereotaxic injections of 3x2 µg <NUM>-OHDA (in <NUM> degree angle) into the left striatum (coordinates relative to bregma and dura A/P +<NUM>; L/M -<NUM>; D/V -<NUM>, A/P <NUM>: L/M -<NUM>; D/V -<NUM> and A/P -<NUM>; L/M: -<NUM>; D/V -<NUM>). Two weeks later, rats were divided to groups based on their amphetamine-induced rotation results (size of the lesion). Thereafter CDNF (<NUM>µg), C-CDNF (equimolar to CDNF <NUM>µg) and N-CDNF (equimolar to CDNF <NUM>µg) were injected intrastriatally to rats using to the same coordinates as the <NUM>-OHDA. In the reference experiment after division of rats into groups, osmotic minipumps were inserted subcutaneously and cannula was placed into lesioned striatum. The minipump delivered MANF4 (i.e. MANF peptide CKGC, see <CIT>), GDNF or vehicle solution into striatum for two weeks after which the minipump and cannula were removed. Inside neurons <NUM>-OHDA has two ways of action that act synergistically: <NUM>) it accumulates in the cytosol and forms free radicals causing oxidative stress; <NUM>) it is a potent inhibitor of the mitochondrial respiratory chain complexes I and IV. Noradrenergic neurons were protected by using a NAT-inhibitor desipramine (<NUM>/kg, i. , <NUM> mins before <NUM>-OHDA -injection). The size of the unilateral lesion and the effect of the treatments were measured with amphetamine induced rotational behavior <NUM>, <NUM>, <NUM> and <NUM> weeks after the lesion in the experiment involving CDNF, C-CDNF, N-CDNF and PBS treated rats, and at <NUM>,<NUM>,<NUM>,<NUM> and <NUM> weeks in the reference experiment with involving MANF4 and GDNF. The number of amphetamine-induced (<NUM>,<NUM>/kg, i. ) full (<NUM>°) ipsi- and contralateral turns were recorded for <NUM> mins after a <NUM> habituation period. The results are expressed as net ipsilateral turns to the lesion side Exclusion criterion was Mean (net rotations) ± <NUM> × STDEV.

Perfusion and tissue processing. Immediately after neurorestoration studies, the rats were anesthetized with an overdose of sodium pentobarbital (<NUM>/kg, i. ; Orion Pharma) and perfused intracardially with PBS followed by <NUM>%paraformaldehyde in a <NUM> sodium phosphate buffer, pH <NUM>. The brains were removed, postfixed for <NUM> and stored in sodium phosphate buffer containing <NUM>% sucrose at <NUM>. Serial coronal frozen sections of <NUM> depth were cut on a sliding microtome. Immunohistochemistry was performed as described elsewhere (Voutilainen et al. , <NUM>), The perfused brains were postfixed overnight in paraformaldehyde at <NUM> and stored in <NUM>% sucrose. The brains were cut into <NUM>-µm-thick sections in series of six. Free-floating sections were washed with phosphate-buffered saline (PBS), and the endogenous peroxidase activity was quenched with <NUM>% hydrogen peroxide (Sigma Aldrich). To block the nonspecific binding of antibodies, the sections were incubated for <NUM> hr in blocking buffer (<NUM>% bovine serum albumin and <NUM>% Triton X-<NUM> in <NUM> × PBS). The sections were incubated overnight in mouse monoclonal anti-tyrosine hydroxylase (TH) antibody (<NUM>:<NUM>,<NUM>; catalog No. MAB318; RRID:AB_2201528; Millipore, Billerica, MA) in blocking buffer at <NUM>, followed by incubation in biotinylated secondary antibody (<NUM>:<NUM>; anti-rat or anti-mouse; Vector, Burlingame, CA). The staining was augmented with avidin-biotin-enzyme complex (ABC kit; Vector), and the signal was visualized with <NUM>',<NUM>'-diaminobenzidine as a chromogen.

TH-positive cells in the substantia nigra pars compacta (SNpc) were analyzed from six sections spanning the SNpc, from approximately A/P -<NUM> to -<NUM> relative to bregma. Cells were counted with a Matlab (RRID:nlx_153890; MathWorks, Natick, MA) algorithm from the images obtained with the 3DHistech scanner. The resolution of the scanner was <NUM>/pixel with a × <NUM> NA <NUM> objective.

The optical densities of the TH-positive neurites in the striatum were determined from three striatal sections, from approximately A/P + <NUM>, + <NUM>, and -<NUM> relative to bregma from each rat. To decrease background signal, the sections were scanned with an automated scanner (3DHistech, Budapest, Hungary, with scanning service provided by the Institute of Biotechnology, University of Helsinki), and the images were converted to <NUM>-bit gray scale. Because the corpus callosum was devoid of TH signal, it was used as a measure of nonspecific background staining. The integrated densities divided by area from the obtained images were analyzed in ImageJ (NIH). Data are presented as percentage of the intact side.

Islets from female, virgin <NUM> weeks old C57bl6Rcc mice were isolated. Islets were recovered o/n in growth medium and the next day equal numbers of islets/well (<NUM>/well) were treated for <NUM> days with placental lactogen (PL <NUM> ng/ml), C-MANF, or MANF. Half of the medium was changed daily to fresh medium with growth factors. Edu, a nucleoside analog alternatively to BrdU (Click-iT®Edu proliferation kit, Invitrogen) was added <NUM> hrs prior to islet harvesting. Islets were broken with trypsin and centrifuged onto glass slides in cytocentrifuge. Cells were fixed after cytospins and proliferating cells stained with Click-iT AlexaFluor azide color reagent, followed by insulin staining (guinea-pig <NUM>:<NUM>, Abcam, Cambridge, UK) o/n at +<NUM> to detect beta cells. Cells were washed and stained with secondary antibodies conjugated with Alexa Fluor® <NUM> (<NUM>:<NUM>, Molecular Probes, Life Technologies, CA, USA). Slides were mounted with Vectashield mounting medium containing DAPI (Vector Laboratories, Inc. , Burlingame, CA, USA). Twelve images (<NUM> x magnification) were acquired with Fluorescence Zeiss AxioImager M2 <NUM> epifluorescence microscope equipped with 40x/Plan-Apochromat/<NUM> Corr M27 and 63x/Plan-Apochromat/<NUM> Oil/M27 and <NUM> AxioCam HRm camera using AxioVision4 software and analysed by Image Pro Plus software (Media Cybernetics, Bethesda, MD, USA) to quantify number of DAPI-positive nuclei. The relative numbers of proliferating beta cells were quantified and compared to wells of three to five repeats/treatment.

Transgenic SOD1 G93A mice served as a transgenic mouse model for ALS in this study. Transgenic mice containing various human SOD1 mutations develop progressive neurodegeneration and motoneuron (MN) death, providing an animal model that has been commonly used for preclinical trials and that has greatly contributed to the understanding of FALS pathogenesis (Gurney et al. Transgenic SOD1 mice exhibit the ALS-like clinical features that are transmitted in an autosomal dominant fashion. In these mice hind limb weakness and tremulous movement appear as initial symptoms at <NUM>-<NUM> week of age, followed by major symptoms such as progressive motor paralysis and neurogenic amyotrophy (Shibata <NUM>). These mice subsequently show disability of gait, eating and drinking and die within some weeks, usually at <NUM>-<NUM> week of age. The transgenic mice carrying the human SOD1 with the glycine93 to alanine mutated were originally obtained from The Jackson Laboratory (http://www. org), Bar Harbor, ME; Strain B6SJL-TgN (SOD1-G93A) 1Gur). Transgenic expression was analyzed by DNA tail tests and PCR, using specific oligonucleotides and conditions as done previously by others (see homepage Jackson Lab). In all the experiments the wild-type B6SJL-TgN (SOD1) 2Gur were included as controls.

In the single administration experiment, mice at around <NUM> weeks of age received a single intracerebroventricular injection of PBS or C-CDNF (<NUM>µg that is equimolar to full-length CDNF <NUM>µg diluted in PBS) under isofluorane anesthesia. Mice were then evaluated for signs of disease and body weight changes twice a week. Evaluation was completed by a battery of behavioral tests designed to assess motor activity in the mice; the battery comprises tests e.

In the chronic infusion experiment, <NUM>-week-old SOD1 mice were inserted a brain infusion cannula (connected via catheter-tubing to an Alzet osmotic minipump) to the right lateral ventricle under isoflurane anesthesia. C-CDNF (<NUM>µg/<NUM>) was infused for <NUM> days. Motor behavior was evaluated with rotarod. Mice were evaluated for clinical signs and body weight changes.

Clinical scoring for SOD1 mice was done using instructions from Jackson laboratory. The mice were carefully examined <NUM> times a week after they were <NUM> weeks old. Animals were scored by lifting them gently by the base of their tails and observing them for tremors, stiffness and their ability to extend their limbs. The clinical scoring is on a scale of <NUM> to <NUM>, based on ALSTDI (ALS therapy Development Institute) hind limb neurological scoring system.

In rotarod, mice were put to a rotating rod (accelerating speed <NUM>-<NUM> rpm/minute), (Ugo Basile, Italy). Cut off time was <NUM> minutes. Rotarod test was done <NUM> times a week after the mice were <NUM> weeks old.

Male Sprague Dawley rats (weight <NUM>-<NUM>, Envigo, Netherlands) were used for the experiments which were conducted according to the 3R principles of EU directive <NUM>/<NUM>/EU on the care and use of experimental animals, local laws and regulations, and were approved by the national Animal Experiment Board of Finland All experiments were performed in a blinded manner and the rats were assigned to different treatment groups randomly. Rats were anesthesized with chloral hydrate (<NUM>/kg, i. A cortical stroke was induced by occluding the distal middle cerebral artery (dMCA) together with a bilateral common carotid artery (CCA) occlusion for <NUM> as described previously (Chen, et al. Briefly, the bilateral CCAs were identified and isolated through a ventral midline cervical incision. Rats were placed in stereotaxic apparatus and a craniotomy was made in the right hemisphere. The right (MCA) was ligated with a <NUM>-<NUM> suture and bilateral common carotids (CCA) were ligated with non-traumatic arterial clamps for <NUM> minutes. After sixty minutes of ischemia, the suture around the MCA and arterial clips on CCAs were removed to introduce a reperfusional injury. After recovery from anesthesia, the rats were returned to their home cage. Body temperatures during and after surgery were maintained at <NUM>.

To test the neuroprotective effect of subcutaneous C-CDNF, <NUM>µg of C-CDNF was given <NUM>-<NUM> before dMCA occlusion and immediately after reperfusion in volume of <NUM>µl s. Phosphate-buffered saline (PBS) was used as vehicle control. The rats were euthanized <NUM> days after dMCAo to measure the infarction volume by <NUM>% <NUM>,<NUM>,<NUM>-triphenyltetrazolium chloride (TTC; Sigma Aldrich, St. Louis, MO) staining. Rats were decapitated and the brains were removed and sliced into <NUM>-mm-thick sections using an acrylic rat brain block. The brain slices were incubated in a <NUM>% TTC solution (Sigma, St. Louis, MO, USA) for <NUM> at room temperature and then transferred into a <NUM>% paraformaldehyde solution for fixation. The area of infarction in each slice was measured with a digital scanner and ImageJ software. The volume of infarction in each animal was obtained from the product of average slice thickness (<NUM>) and sum of infarction areas in rostral brain slices examined. Student's t-test was used for statistical analysis.

C-CDNF peptides with N-terminal and/or C-terminal deletions (see Tables <NUM> and <NUM>) were produced by custom peptide synthesis (Stawikowski and Fields, Curr Protoc Protein Sci. ; <NUM> February CHAPTER: Unit-<NUM>. Bioassays as described above were performed with each of the peptides. The results are shown in Tables <NUM>-<NUM> and <FIG>.

Protease cleavage site prediction of C-terminal CDNF fragment and C-terminal MANF fragment was performed with PROSPER software at https://prosper. Predicted protease cleavage sites are shown in Tables <NUM> and <NUM> and in <FIG> and <FIG>.

A library of fragments, from C-terminal CDNF fragments and/or C-terminal MANF fragments, can be designed based on the information from protease cleavage database, for example, using MEROPS database at https://www. uk/merops/index. shtml and substituting one or more of the native amino acids, for example, at positions P4-P3-P2-P1-P1' or P2-P1-P1', with amino acids that are used less, are known to inhibit the protease or are modified. The fragment library may be synthesized in a <NUM>-well or <NUM>-well format, for example. The fragment for protease assay may consist of, for example, <NUM>-<NUM> amino acids (depending on the protease and the assay format) wherein the C-terminal amino acid is linked to a fluorescent dye, for example, <NUM>-amino-<NUM>-methylcoumarin or amino-<NUM>-trifluoromethyl (AMC or AFC). Designs of the fragments may be: P6-P5-P4-P3-P2-P1, P5-P4-P3-P2-P1-, P4-P3-P2-P1, P3-P2-P1-, or P2-P1- combined with P1'-P2'-P3'-P4'-P5'-P6'-AMC, P1'-P2'-P3'-P4'-P5'-AMC, P1'-P2'-P3'-P4'-AMC, P1'-P2'-P3'-AMC, P1'-P2'-AMC, or P1'-AMC, wherein amino acid residues are consecutively numbered outward from the cleavage site and the scissile bond is located between the P1 and P1' positions. Recombinant Human MMP-<NUM>, MMP-<NUM>, MMP-<NUM>, and elastase-<NUM> proteins are purchased, for example, from R&D Systems. To test and activate the protease, instruction given by the manufacturer are followed. Synthesized fragments are dissolved in appropriate buffer to an appropriate concentration. Protease reactions are performed in reaction mixtures containing the fragment and the protease in the protease-specific buffer. Reactions are carried out at +<NUM> and aliquots of the reaction mixture are taken at different time points to determine protease cleavage activity. Fragments are analysed, for example, with MALDI-MS or HPLC and protease cleavage rates are determined.

Claim 1:
A C-terminal CDNF fragment with a length of <NUM>-<NUM> amino acids comprising at least the consecutive amino acid residues at positions <NUM>-<NUM> or <NUM>-<NUM> of the sequence as set forth in SEQ ID NO:<NUM>:
<IMG>
or a sequence which has at least <NUM> % sequence identity with the sequence of positions <NUM>-<NUM> or <NUM>-<NUM> in SEQ ID NO:<NUM>, wherein said fragment is a cell membrane penetrating peptide, preferably capable of penetrating human blood-brain barrier, and has a protective effect on neuronal cells, for use as a medicine.